Compositions and Methods for Detecting and Treating Brain Injury Associated Fatigue and Altered Cognition (BIAFAC)

ABSTRACT

The present invention includes composition, methods and kits for detecting and treating brain injury associated fatigue or altered cognition (BIAFAC) in a human patient comprising: identifying a human patient in need of treatment for brain injury associated fatigue or altered cognition associated with an altered intestinal flora; and providing the human patient with a composition comprising at least one of: a Prevotella spp or a Bacteroidies spp bacteria, or one or more agents that increase the amount of the Prevotella spp or the Bacteroidies spp bacteria in an intestinal flora of the human patient to reduce or eliminate the brain injury associated fatigue or altered cognition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 16/449,047, now allowed, filed on Jun. 21, 2019 and claimspriority to U.S. Provisional Application Ser. No. 62/688,106, filed Jun.21, 2018, the entire contents of which are incorporated herein byreference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

Not Applicable.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of novelcompositions and methods for detecting and treating brain injuryassociated fatigue and altered cognition (BIAFAC).

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with traumatic brain injury and changes in cognition.

Following traumatic brain injury (TBI) or other central nervous system(CNS) maladies (e.g. stroke or hemorrhage), patients develop a clinicalsyndrome characterized by fatigue, altered cognition, chronicinflammation and altered amino acid absorption. Unfortunately, currenttreatments for these conditions (referred to herein as brain injuryassociated fatigue and altered cognition (BIAFAC)) involve dailyinjection of expensive recombinant human growth hormone that treats thesyndrome but does not produce a lasting cure.

Others have attempted to treat TBI or other CNS maladies with varyingresults. One such patent is International Publication No. WO2017152137A3, filed by Lynch, et al., entitled “Microbial consortium and usesthereof”. These applicants teach microbial compositions and methods ofusing the same that include therapeutically effective amounts ofLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus, which aresaid to be particularly useful for methods of treating and preventinginflammatory diseases.

Another such patent is International Publication No. WO2017191638A1,filed by Ilan, entitled “Hyperimmune colostrum in the modulation andtreatment of conditions associated with the mammalian microbiome”. Thisinventor is said to teach methods and compositions for modulating,preventing or treating non-clinical and clinical conditions that areassociated with a mammalian microbiome. The methods and compositions aresaid to include preparations of hyperimmune colostrum enriched withantibodies for lipopolysaccharides (anti-LPS) that directly orindirectly impact the mammalian microbiome to alleviate and treatvarious forms of renal failure, hypertension, heart disease,atherosclerosis and sepsis, and various psychiatric and neurobehavioralconditions.

Another such patent is International Publication No. WO2017181158A1,filed by Costa-Mattioli, et al., entitled “Probiotic therapies fordevelopmental disorders and other neurological disorders”. Theseinventors teach methods and compositions to manipulate the microbiome inan individual having at least one social behavior deficit. Moreparticularly, an individual that has at least one social behaviordeficit and was born from a mother who during pregnancy was obese,overweight, or on a high-fat diet during pregnancy or carries mutationsassociated with neurodevelopmental disorders is provided an effectiveamount of Lactobacillus reuteri for the improvement of at least onesymptom of a social behavior deficit.

Finally, another patent is U.S. Patent Publication No. 20120128711,filed by Hausman, et al., that is entitled “Anti-inflammatory approachto prevention and suppression of post-traumatic stress disorder,traumatic brain injury, depression and associated disease states”. Thisapplication is said to teach composition and methods for the preventionand control of inflammation and oxidative stress and various associatedmedical conditions, including PTSD, chronic depression and traumaticbrain injury. These applicants disclose a composition that includes aphytonutrient and enriched mushrooms having enhanced Vitamin D andergothioneine. The enriched mushroom and phytonutrient are said toprovide a synergistic effect on cellular longevity and/or cellularrejuvenation of subjects with both a normal and nutritionally deficientdiets, improved tolerance to oxidative and/or inflammatory stress as aresult of the neutralization of free radicals and prevention of chronicinflammation.

However, a need remains for tests and treatments for brain injuryassociated fatigue and altered cognition resulting for diseases orconditions that cause decreased cognition.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of treatingbrain injury associated fatigue or altered cognition (BIAFAC) in a humanpatient comprising: identifying a human patient in need of treatment forbrain injury associated fatigue or altered cognition associated with analtered intestinal flora; and providing the human patient with acomposition comprising at least one of: a Prevotella spp or aBacteroidies spp bacteria, or one or more agents that increase theamount of the Prevotella spp or the Bacteroidies spp bacteria in anintestinal flora of the human patient to reduce or eliminate the braininjury associated fatigue or altered cognition. In one aspect, the humanpatient has brain injury that is chronic, mild, or undiagnosed. Inanother aspect, the human patient has brain injury caused by stroke,hemorrhage, surgery, or radiation. In another aspect, the compositionfurther comprises an amino acid mixture that promotes the growth ofPrevotella spp or Bacteroidies spp bacteria. In another aspect, thecomposition further comprises an agent that reduces or eliminatesRuminococcaceae genus bacteria. In another aspect, the agent thatreduces or eliminates Ruminococcaceae genus bacteria is selected from anamino acid mixture, an antibacterial agent, a bacteriophage, or anantimicrobial CRISP-Cas system agent. In another aspect, the humanpatient did not receive a concurrent antibiotic or a probiotic therapy.In another aspect, the bacteria is listed are responsive in Tables 3, 4,5 and 6. In another aspect, the method further comprises providing thepatient with a fecal transplant comprising Prevotella spp orBacteroidies spp bacteria or healthy bacterial community containingPrevotella spp or Bacteroidies spp. In one aspect, the method furthercomprises providing the human patient a bacterial composition that ismodified to correct a TBI flora to a normal flora is provided before,during, or after undergoing a colon cleanse treatment, delivered bycolonoscope, wherein the bacterial composition is not a fecaltransplant, or wherein bacteria are lab-grown, or wherein bacteria arelab-grown and customized to specifically modify a TBI flora of aspecific patient.

In another embodiment, the present invention includes a method ofidentifying a patient with a brain injury associated fatigue or alteredcognition (BIAFAC) that will benefit from a probiotic bacteria treatmentcomprising: identifying a human patient with a brain injury associatedfatigue or altered cognition; obtaining a biological sample from thepatient that comprises gut intestinal flora; and determining whether thegut intestinal flora in the biological sample comprises a decrease inthe presence of probiotic bacteria selected from at least one ofPrevotella spp or Bacteroidies spp bacteria, when compared to a normalhuman sample. In one aspect, the method further comprises treating thesubject with a composition comprising one or more probiotic bacteriaselected from at least one of: Prevotella spp or Bacteroidies spp, orone or more agents that increase the amount of Prevotella spp orBacteroidies spp in an intestinal flora of the patient, wherein thecomposition comprises an amount effective to reduce or eliminate thebrain injury associated fatigue or altered cognition. In another aspect,the method further comprises providing the patient with a fecaltransplant comprising Prevotella spp or Bacteroidies spp. In anotheraspect, the biological sample is a fecal sample. In another aspect, thepresence of the probiotic bacteria is determined by at least one ofmetagenomic shotgun sequencing of bacteria, quantitative PCR assays ormetabolic LC-MS analysis of bacterial metabolites. In another aspect,the method further comprises the step of providing the patient with anagent that reduces or eliminates Ruminococcaceae genus bacteria. Inanother aspect, the agent that reduces or eliminates Ruminococcaceaegenus bacteria is selected from an amino acid mixture, an antibacterialagent, a bacteriophage, or an antimicrobial CRISP-Cas system agent. Inanother aspect, the human patient has brain injury that is chronic,mild, or undiagnosed. In another aspect, the human patient has braininjury caused by stroke, hemorrhage, surgery, or radiation. In anotheraspect, the composition further comprises an amino acid mixture thatpromotes the growth of Prevotella spp or Bacteroidies spp. In anotheraspect, the composition further comprises an amino acid mixture thatreduces or eliminates Ruminococcaceae genus bacteria. In another aspect,the patient did not receive a concurrent antibiotic or a probiotictherapy.

In another aspect, one or more bacteria for treatments are listed inTables 3, 4, 5 and/or 6. In another aspect, a bacterial composition thatis modified to correct a TBI flora to a normal flora is provided before,during, or after undergoing a colon cleanse treatment, delivered bycolonoscope, wherein the bacterial composition is not a fecaltransplant, or wherein bacteria are lab-grown, or wherein bacteria arelab-grown and customized to specifically modify a TBI flora of aspecific patient.

In another embodiment, the present invention includes a composition fortreating brain injury associated fatigue or altered cognition in a humanpatient comprising one or more probiotic bacteria selected from at leastone of: Prevotella spp or Bacteroidies spp, in an effective amountsufficient to reduce or eliminate the brain injury associated fatigue oraltered cognition. In one aspect, the composition further comprises afecal transplant comprising Prevotella spp or Bacteroidies spp. Inanother aspect, the composition further comprises an agent that reducesor eliminates Ruminococcaceae genus bacteria. In another aspect, theagent that reduces or eliminates Ruminococcaceae genus bacteria isselected from an amino acid mixture, an antibacterial agent, abacteriophage, or an antimicrobial CRISP-Cas system agent. In anotheraspect, the composition further comprises an amino acid mixture thatpromotes the growth of Prevotella spp or Bacteroidies spp. In anotheraspect, the patient did not receive a concurrent antibiotic or aprobiotic therapy. In another aspect, one or more bacteria fortreatments are listed in Tables 3, 4, 5 and/or 6. In another aspect, abacterial composition that is modified to correct a TBI flora to anormal flora is provided before, during, or after undergoing a coloncleanse treatment, delivered by colonoscope, wherein the bacterialcomposition is not a fecal transplant, or wherein bacteria arelab-grown, or wherein bacteria are lab-grown and customized tospecifically modify a TBI flora of a specific patient.

In another embodiment, the present invention includes a blood test assaythat determines a biological profile associated with a bacterial florathat exacerbates a brain injury associated fatigue or altered cognitionfor testing a fecal sample from a patient that compares the biologicalprofile from a patient to a database of a normal biological profile,wherein the bacterial flora detected is selected from Prevotella spp,Bacteroidies sp, or Ruminococcaceae genus bacteria. In one aspect, thebiological profile is further defined as comprising an array thatspecifically tests for an amino acid profile associated with thepresence, absence, or amount of the Prevotella spp, Bacteroidies sp, orRuminococcaceae genus bacteria. In another aspect, the biologicalprofile is further defined as comprising an array that specificallytests for a nucleic acid profile associated with the presence, absence,or amount of the Prevotella spp, Bacteroidies sp, or Ruminococcaceaegenus bacteria. In another aspect, the patient did not receive aconcurrent antibiotic or a probiotic therapy. In another aspect, one ormore bacteria are listed in Tables 3, 4, 5 and/or 6.

In another embodiment, the present invention includes a kit forscreening a patient for a brain injury associated fatigue or alteredcognition and choosing a proper treatment based on the screening, thekit comprising: a screening tool that screens the patient for a risk ofbrain injury associated fatigue or altered cognition, the screening toolcomprising a questionnaire and further comprising a screening scoresystem that determines the risk of the patient based on a cumulativepoint total from answers to the questionnaire; a diagnostic test thatcategorizes the brain injury associated fatigue or altered cognitionbased on the cumulative point total from the questionnaire; a fecalsample collection container for determining a bacterial flora in a fecalsample of the patient; instructions for implementing the brain injuryassociated fatigue or altered cognition treatment; and a brain injuryassociated fatigue or altered cognition treatment composition comprisingone or more probiotic bacteria selected from at least one of: Prevotellaspp or Bacteroidies spp, or one or more agents that increase the amountof Prevotella spp or Bacteroidies spp in an intestinal flora of thepatient, wherein the composition comprises an amount effective to reduceor eliminate the brain injury associated fatigue or altered cognition.In one aspect, the bacterial flora detected is a presence or an amountof a Prevotella spp, Bacteroidies spp or Ruminococcaceae genus bacteriain the fecal sample. In another aspect, the kit comprises a containerwith reagents for detecting at a genus or species level the bacterialflora selected from at least one of Ion Torrent Personal Genome Machine(PGM), next-generation sequencing (NGS), or qualitative Polymerase ChainReaction (qPCR). In another aspect, the brain injury associated fatigueor altered cognition treatment is a probiotic therapy regime of thepatient based the patient's brain injury associated fatigue or alteredcognition. In another aspect, the probiotic therapy regime furthercomprises one or more agents that reduces or eliminates Ruminococcaceaegenus bacteria. In another aspect, the human patient has a brain injurythat is chronic, mild, undiagnosed. In another aspect, the human patienthas a brain injury caused by stroke, hemorrhage, surgery, or radiation.In another aspect, the composition further comprises a fecal transplantcomprising Prevotella spp or Bacteroidies spp. In another aspect, thecomposition further comprises an amino acid mixture that promotes thegrowth of Prevotella spp or Bacteroidies spp. In another aspect, thecomposition further comprises an amino acid mixture that reduces oreliminates Ruminococcaceae genus bacteria. In another aspect, thecomposition further comprises an agent that reduces or eliminatesRuminococcaceae genus bacteria.

In another aspect, the agent that reduces or eliminates Ruminococcaceaegenus bacteria is selected from an antibacterial agent, a bacteriophage,an antimicrobial CRISP-Cas system agent. In another aspect, the patientdid not receive a concurrent antibiotic or a probiotic therapy. Inanother aspect, one or more bacteria for treatments are listed in Tables3, 4, 5 and/or 6. In another aspect, a bacterial composition that ismodified to correct a TBI flora to a normal flora is provided before,during, or after undergoing a colon cleanse treatment, delivered bycolonoscope, wherein the bacterial composition is not a fecaltransplant, or wherein bacteria are lab-grown, or wherein bacteria arelab-grown and customized to specifically modify a TBI flora of aspecific patient.

In another embodiment, the present invention includes a method ofdiagnosing and treating a patient for brain injury associated fatigue oraltered cognition, the method comprising: using an algorithm implementedin a computer program to: screen a patient for a brain injury associatedfatigue or altered cognition risk by scoring a questionnaire based onanswers from the patient to the questionnaire, the scoring providing acumulative point total, and determining the brain injury associatedfatigue or altered cognition of the patient based on the cumulativepoint total from the questionnaire; and choosing a proper brain injuryassociated fatigue or altered cognition treatment product if the braininjury associated fatigue or altered cognition risk exceeds a threshold,the brain injury associated fatigue or altered cognition treatmentproduct comprising a composition comprising one or more probioticbacteria selected from at least one of: Prevotella spp or Bacteroidiesspp, or one or more agents that increase the amount of Prevotella spp orBacteroidies spp in an intestinal flora of the patient, wherein thecomposition comprises an amount effective to reduce or eliminate thebrain injury associated fatigue or altered cognition. In another aspect,the patient preparation instructions include following dietarymodifications in accordance with brain injury associated fatigue oraltered cognition treatment specifications to at least one of: increasethe amount of Prevotella spp or Bacteroidies spp in the intestinal floraof the patient, or reduces or eliminates Ruminococcaceae genus bacteriain the intestinal flora of the patient. In another aspect, the patientdid not receive a concurrent antibiotic or a probiotic therapy. Inanother aspect, one or more bacteria for treatments are listed in Tables3, 4, 5 and/or 6. In another aspect, a bacterial composition that ismodified to correct a TBI flora to a normal flora is provided before,during, or after undergoing a colon cleanse treatment, delivered bycolonoscope, wherein the bacterial composition is not a fecaltransplant, or wherein bacteria are lab-grown, or wherein bacteria arelab-grown and customized to specifically modify a TBI flora of aspecific patient, or one or more bacteria listed in Tables 3, 4, 5, or 6as being different from a patient with a brain injury when compared to anormal patient.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A and 1B show a comparison of fecal microbiome communitystructure of TBI and control cohorts determined by 16S V4 NGS. Weighted(FIG. 1A) and unweighted (FIG. 1B) UniFrac principal component analyses(PCoA) were completed to determine if the microbiome community profileswere different between the cohorts. Weighted analysis showed significantdifferences when considering relative abundance (p=0.002). Unweightedcomparisons for the presence or absence of bacterial OTUs alsoestablished significantly different profile (p=0.005).

FIG. 2 shows the Alpha and Shannon diversity index (SDI) comparisonsbetween TBI and control cohort community profiles. Overall numbers ofOTUs were not statistically different (p=0.21) as shown by alphadiversity calculations were completed using R³⁰. The SDI wassignificantly higher in TBI cohorts compared to controls (p=0.008).Alpha diversity differences between TBI and control datasets wereassessed by Wilcoxon rank-sum tests. Evaluation of beta diversity(Unifrac distance) was completed by. PERMANOVA. All P-values wereadjusted for multiple comparisons with the FDR algorithm³¹.

FIGS. 3A and 3B show a comparison of the average relative abundance ofindicated OTUs detected in TBI and control fecal bacterial communities.The plots illustrate individual levels for each indicated target(scatter plot) as well as the mean, standard deviations and 95%confidence intervals (box and whiskers). FIG. 3A shows differences inthe relative abundance of the indicated phyla. FIG. 3B illustratesoutcomes for selected families. Significant differences are indicated byasterisks and detailed in the results section.

FIGS. 4A to 4C show the significant differences in absolute abundanceestablished by customized fecal microbiome qPCR array. Using a novel 96target qPCR array developed for this study, absolute abundance for 94selected bacterial targets and 2 controls (16S rRNA gene and human GAPDHgene) supported clustering analysis using Morpheus web-based software(Broad Institute, Cambridge, Mass.) was completed on individual TBI andcontrol profiles as shown in FIG. 4A. Statistically significantdifferences in the average qPCR absolute abundance data was determinedusing multiple t tests via the Holm-Sidak method (GraphPad Prism,v.7.0e). FIGS. 4B and 4C show the average abundance and standard errorfor each selected target as indicated in the results section. Nodifferences were observed in 16S abundance or human genomic DNA content.Significant differences are indicated by the asterisks. Detailed dataare provided in Table 2.

FIG. 5 shows a heat map associating amino acid concentrations withbacterial relative abundance. Using MixOmics³² implemented in R[v3.3;³⁰] correlations between the bacterial relative abundance and theconcentration of selected amino acids was completed using sparse partialleast squares regression (sPLS) performed in canonical mode with LASSOpenalization. The results for 2 Ruminococcaceae spp. and Prevotella spp.are presented as a heat map.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below.

Terms defined herein have meanings as commonly understood by a person ofordinary skill in the areas relevant to the present invention. Termssuch as “a”, “an” and “the” are not intended to refer to only a singularentity, but include the general class of which a specific example may beused for illustration. The terminology herein is used to describespecific embodiments of the invention, but their usage does not limitthe invention, except as outlined in the claims.

The present invention provides, for the first time, a direct linkbetween altered cognition/fatigue experienced by patients with atraumatic brain injury (TBI) and the gastrointestinal microbiome. Thisaltered cognition or syndrome is referred to as Brain Injury AssociatedFatigue and Altered Cognition (BIAFAC), as it was found to includeshifted bacterial communities in fecal materials. It was found thatpatients, who had suffered a TBI when compared to a control group, had asignificantly altered microbiome that was associated with the reducedcognition.

The present inventors identified that the microbiome of patients withBIAFAC lacks Prevotella spp and Bacteroidies spp. Conversely, bacteriain the Ruminococcaceae genus are increased in abundance compared tohealthy control samples. This work identified specific organisms by bothnext generation sequencing and quantitative PCR approaches that form thebasis of both diagnostic and therapeutic inventions described herein.The inventors identified novel, therapeutically relevant biomarkers thatcan be used to treat symptomatic, chronic TBI patients, and offeringclinically meaningful treatment options for TBI-related comorbidities.Given the complexity of the impact on both the brain, CNS, immune,metabolic, inflammatory, pituitary, and intestinal microbiome, theresults herein yielded both therapeutic and mechanistic insights intoTBI. The result herein show that supplementation or replacement of thedysbiotic intestinal community via, e.g., fecal microbiota transplant(FMT) can be used to treat TBI patients and its associatedcomorbidities.

For enhanced diagnostics, the present invention used a qPCR array todiagnose individuals with BIAFAC-associated gastrointestinal (GI)microbiome changes and to monitor the success of therapeutic approaches.Another such array uses customized qPCR assays assembled into kittedsystems for use in clinical microbiology facilities. The array can beused to identify optimal donated fecal material from healthy donors tosupport development of therapeutic microbiome transplants. Thetherapeutic composition that include oral compositions, suppositoriesand/or enemas that include supplementation with identified and culturedprobiotic organisms reduced or lost in the GI microbiome of BIAFACpatients.

The probiotic bacteria, prebiotic agents, and/or xenobiotics for usewith the present invention can be provided in a variety of dosage forms.For example, e.g., tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, enemas, suppositories, and emulsions maybe used to provide the probiotic bacteria, prebiotic agents, and/orxenobiotics of the present invention to a patient in need of therapy forbrain injury associated fatigue and/or altered cognition.

Techniques and compositions for making useful dosage forms using thepresent invention are described in one or more of the followingreferences: Anderson, Philip O.; Knoben, James E.; Troutman, William G,eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002;Pratt and Taylor, eds., Principles of Drug Action, Third Edition,Churchill Livingston, N.Y., 1990; Katzung, ed., Basic and ClinicalPharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman,eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGrawHill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., LippincottWilliams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia,Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all ofwhich are incorporated by reference, and the like, relevant portionsincorporated herein by reference.

For example, the probiotic bacteria, prebiotic agents, and/orxenobiotics may be included in a tablet. Tablets may contain, e.g.,suitable binders, lubricants, disintegrating agents, coloring agents,flavoring agents, flow-inducing agents and/or melting agents. Forexample, oral administration may be in a dosage unit form of a tablet,gelcap, caplet or capsule, the active drug component being combined witha non-toxic, pharmaceutically acceptable, inert carrier such as lactose,gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesiumstearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol,mixtures thereof, and the like. Suitable binders for use with thepresent invention include: starch, gelatin, natural sugars (e.g.,glucose or beta-lactose), corn sweeteners, natural and synthetic gums(e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants for use with theinvention may include: sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride, mixturesthereof, and the like. Disintegrators may include: starch, methylcellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.

In one embodiment, gelatin capsules (gelcaps) may include the probioticbacteria, prebiotic agents, and/or xenobiotics, and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Like diluents may be used to make compressedtablets. Both tablets and capsules may be manufactured asimmediate-release, mixed-release or sustained-release formulations toprovide for a range of release of medication over a period of minutes tohours. Compressed tablets may be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere. An entericcoating may be used to provide selective disintegration in, e.g., thegastrointestinal tract.

For oral administration in a liquid dosage form, the probiotic bacteria,prebiotic agents, and/or xenobiotics may be adapted for oraladministration. Examples of suitable liquid dosage forms includepowders, tablets, gelcaps, solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, emulsifying agents, suspending agents, diluents, sweeteners,thickeners, and melting agents, mixtures thereof, and the like, that donot affect the viability of the probiotic bacteria or enhance theviability of the probiotic bacteria.

Liquid dosage forms for oral administration may also include coloringand flavoring agents that increase patient acceptance and thereforecompliance with a dosing regimen. In general, water, a suitable oil,saline, aqueous dextrose (e.g., glucose, lactose and related sugarsolutions) and glycols (e.g., propylene glycol or polyethylene glycols)may be used as suitable carriers for parenteral solutions. Solutions forparenteral administration include generally, a water soluble salt of theactive ingredient, suitable stabilizing agents, and if necessary,buffering salts. Antioxidizing agents such as sodium bisulfite, sodiumsulfite and/or ascorbic acid, either alone or in combination, aresuitable stabilizing agents. Citric acid and its salts and sodium EDTAmay also be included to increase stability. Suitable pharmaceuticalcarriers are described in REMINGTON'S PHARMACEUTICAL SCIENCES, MackPublishing Company, a standard reference text in this field, relevantportions incorporated herein by reference.

Patients with chronic traumatic brain injury (TBI) requiring long-term,permanent care suffer a myriad of clinical symptoms (e.g., impairedcognition, fatigue, and other conditions) that persist for many yearsbeyond the acute brain injury. In addition to these comorbid clinicalsymptoms, chronic TBI patients also exhibit altered amino acid andhormonal profiles with distinct cytokine patterns suggestive of chronicinflammation. The present inventors recognized that this metabolic linkcould involve a role for the gut-brain axis in chronic TBI. Thus, theinventors utilized a two-site trial to investigate the role of thegut-brain axis in the comorbidities of chronic TBI. The fecal microbiomeprofile of 22 moderate/severe TBI patients residing in permanent carefacilities in Texas and California was compared to 18 healthyage-matched control subjects working within the participatingfacilities. Each fecal microbiome was characterized by 16S(V4) rRNA genesequencing and metagenomic genome sequencing approaches followed byconfirmatory full 16S rRNA gene sequencing or focused tuf genespeciation and specific qPCR evaluation of selected genera or species.The average chronic TBI patient fecal microbiome structure wassignificantly different compared to the control cohort, and thesedifferences persisted after group stratification analysis to identifyany unexpected confounders. Notably, the fecal microbiome of the chronicTBI cohort had absent or reduced Prevotella spp and Bacteroidies spp.Conversely, bacteria in the Ruminococcaceae family were higher inabundance in TBI compared to control profiles. Although the sequelae ofgut-brain axis disruption following TBI is not fully understood,characterizing TBI-related alterations in the fecal microbiome providesbiomarkers and therapeutic targets to address patient morbidity.

Traumatic brain injury (TBI) and its accompanying chronic morbiditiesaffects more than 2.5 million individuals annually in the UnitedStates¹. Concern about the long-term effects of brain injury has grownrecently with increased publicity of sport-related chronic traumaticencephalopathy² and military blast-related injury³. TBI is a complexneurologic insult that can lead to chronic, progressive deterioration ofpatient health and increased morbidity. During convalescence,TBI-associated pathologies can advance to full disability aftersignificant periods of relative health and normalcy. In the case ofchronic TBI, where the severity of the injury often requires residencein a long-term care facility, the clinical symptoms are often complexand varied ranging from cognitive deficiencies to fatigue. In thispatient population, supportive treatment is standard of care andadditional understanding is needed to open new therapeutic avenues.

The sequelae of chronic TBI are not well understood. Aside from therecognized pituitary dysfunction that occurs in some patients, thecause(s) of the myriad of other long-term clinical symptoms and theirmediators are uncertain. In the inventors' prior work, chronic TBIpatients were found to exhibit abnormal metabolic responses and alteredrelationships between circulating amino acids, cytokines, and hormones².The present inventors that the gut-brain axis may be linked toneuropathologies⁴⁻⁷.

Notably, the inventors recognized the interactions between thegastrointestinal (GI) tract, immune system and brain has led to a numberof discoveries suggesting that the GI microbiome is a major influence oneach of the systems⁸⁻¹⁰. Further, recent next generation sequencing(NGS) analyses have associated altered GI microbiomes with a growinglist of neuropathologies including Parkinson's disease, autism,Guillain-Barre syndrome, anxiety and depression¹¹. The GI microbiome isone of the most diverse communities of bacteria in the human body withestimates of up to 1,000 distinct species included in typicalcommunities^(12,13). Current understanding is largely based on fecalanalyses by NGS of the variable regions of the bacterial 16S rRNA geneor metagenomic analyses¹³. The large datasets created from variouscohorts have clearly established that this community of bacteriacontributes to basic physiology, immunity and inflammation. Morerecently, dysbiosis has been associated with altered cognition, behaviorand even mood disorders⁴⁻⁶.

Mild TBI is associated with alterations in gut metabolism soon afterinjury with direct impacts upon the intestinal mucosa including loss oftight junctions that contribute to increased intestinal permeability,inflammation and malabsorption ^(14,15). Patients with mild TBI can alsodevelop sequelae years after injury presenting with profound fatigue andaltered cognition. By way of explanation, and not a limitation of thepresent invention, the inventors recognized that mucosal alterationslikely foster dysbiotic conditions to which the intestinal microbiotaadapt ultimately establishing an altered bacterial population thatpropagates the sequelae.

The present inventors determined whether the fecal microbiome is alteredin chronic, moderate-severe TBI patients in permanent care facilitiescompared to healthy control subjects working at the facilities. Theinventors further determined if TBI-induced dysbiosis indeed disruptsthe gut/brain axis. It was found that the fecal microbiome provides bothbiomarkers and therapeutic targets to address additional sequelaeobserved in chronic TBI patients.

Ethics statement. All patients provided informed consent before blooddrawing or collection of a fecal sample was attempted. All procedureswere approved by the associated IRBs contracted by the Tideway facilityat the Transitional Learning Center (TLC Galveston, Tex.) and Centre forNeuro Skills (CNS; Bakersfield, Calif.). Samples were assigned a uniquestudy number to minimize exposure of personal information.

Patients. Study participants were enrolled from the Tideway TLC facilityin Galveston, Tex. or the CNS facility in Bakersfield, Calif. Twenty-twoparticipants with moderate to severe TBI were recruited to provide fecalsamples. Eighteen individuals were recruited to the control cohortcomposed primarily of facility workers who shared both environment andsome meals with the TBI patients. After informed consent was obtained,participants were fed a standardized meal and blood was collected 90minutes later for serum amino acid analysis.

Participants were provided fecal sampling kits and were instructed onsampling technique. Fecal samples were collected as close to ingestionof the study meal as possible. In some cases, fecal sampling wascompleted by caregivers on behalf of the TBI participants. Thedemographics and details of each cohort are provided in Table 1.

Microbiome analysis and DNA preparation. Preparation of DNA. Fecalsamples were shipped to the Baylor College of Medicine's Alkek Centerfor Metagenomics and Microbiome Research facility on dry ice after beingfrozen and stored at −80° C. in the stabilizing chemical mixtureprovided in the DNA Genotek OMNIgene GUT kit (Genotek, Ottawa, Canada).Following an initial thaw of the material, cell lysing and DNAextraction were performed using the Qiagen processing protocol(described in detail for Illumina NGS below) recommended by theNIH-Human Microbiome Project ^(16,17). At UTMB, a second DNA extractionwas performed on the original fecal material (described in detail belowfor Ion Torrent full 16S rRNA gene NGS or subsequent qPCR analyses). Forboth approaches, DNA quality was assessed for total bacterial contentand human genomic material via qPCR.

16S rRNA gene sequencing and compositional analysis. 16S rRNA genesequencing methods were adapted from the methods developed by theNIH-Human Microbiome Project^(16,17). Bacterial DNA was extracted usingthe PowerMag Microbiome DNA isolation kit following the manufacturer'sinstructions. The V4 region of the 16S rRNA gene was amplified by PCRand sequenced using barcoded Illumina adapter-containing primers 515Fand 806R ¹⁸ on the MiSeq platform (Illumina) using the 2×250 bppaired-end read protocol yielding pair-end reads that overlap almostcompletely. Sequencing read pairs were demultiplexed based on the uniquemolecular barcodes, and reads were merged using USEARCH v7.0.1090¹⁹.Merging allowed zero mismatches with a minimum overlap of 50 bases, andmerged reads were trimmed at the first base with a Q≤5. In addition, aquality filter was applied to the resulting merged reads and thosecontaining above 0.05% expected errors were discarded. Sequences werestepwise clustered into operational taxonomic units (OTUs) at asimilarity cutoff value of 97% using the UPARSE algorithm ²⁰. Chimeraswere removed using USEARCH v7.0.1090. OTUs were determined by mappingthe centroids to the SILVA database ²¹ containing only the 16S rRNA geneV4 region to determine taxonomies. A custom script constructed ararefied OTU table (rarefaction was performed at only one sequencedepth) from the output files generated in the previous two steps fordownstream analyses. The inventors utilized multiple quality controlmeasures, including the use of non-template controls, at the microbialDNA extraction, 16S rRNA gene amplification, and amplicon sequencingprocesses. Resulting OTU tables were rarified to 5,953 reads per sample.

Metagenomic shotgun sequencing and analysis. Individual librariesconstructed from each sample were sequenced using the 2×100 bppaired-end read protocol on the HiSeq platform (Illumina). The processof quality filtering, trimming and demultiplexing was performed using apipeline developed at the Baylor College of Medicine that employs anumber of publicly available tools such as Casava v.8.3 (Illumina) forthe generation of fastqs, Trim Galore and cutadapt ²² for adapter andquality trimming, and PRINSEQ ²³ for sample demultiplexing.Additionally, Bowtie 2 v2.2.1 ²⁴ was used to map reads to customdatabases for bacteria, viruses, human, and vectors and removenon-bacterial reads from the dataset. For bacterial reads, the highestidentity match was chosen. If there were multiple top hits, the lowestcommon ancestor was determined, but these reads did not contribute tothe analysis. Reads whose genomic coordinates overlapped with known KEGGorthologs (KOs) were tabulated. Coding sequences from reference genomesthat have not been specifically annotated by KEGG were aligned to allknown KOs. Any coding sequence that had >70% identity and >70% querycoverage to a known KO was assigned to that KO. This process in effectcreated links between new genomes and the KEGG database. KEGG modules (Mnumbers) were calculated step-wise and determined to be complete if 65%of the reaction steps were present per detected species and for themetagenome as a whole. Pathways were constructed for each taxa andmetagenome by calculating the minimum set through MinPath ²⁵ resultingfrom the gene orthologs present. The number of reads matching a KEGGmodule was averaged across the TBI or control cohorts and compared(unpaired Student's T test) to identify modules that were significantlyover or under represented by the TBI microbiome profiles.

Ion torrent 16S rRNA gene NGS and qPCR evaluations. To confirm the 16SrRNA gene NGS, the second preparation of DNA was analyzed by Ion Torrent16S NGS and qPCR at UTMB for selected organisms. This second DNApreparation addressed differences in recovered DNA species created bydifferent kits as well as the age of the previously extracted material.For the second extraction, after thawing the original material preservedin the OmniGene kit solution, suspended fecal matter was diluted 1:1 inRNeasy PowerMicrobiome kit-provided lysis solution was incubated at 55°C. for 30 minutes (Qiagen, Germantown, Md.) and then subjected to beadbeating utilizing 0.1 mm glass bead-based (Qiagen) homogenization (5minutes at 30 Hz) in a TissueLyser LT (Qiagen). Clarified liquidfractions (13,000×g for 1 minute) were mixed with IRS solution (Qiagen)and incubated at 4° C. for 5 minutes prior to final extraction of DNAusing a MagNA Pure 96 automated nucleic acid extraction DNA and viral NAsmall volume kit (Roche Applied Science, Indianapolis, Ind.).

Ion Torrent fecal microbiome sequencing was carried out using afusion-PCR method. Briefly, fusion-primers were designed in accordancewith the manufacturer's guidelines (Ion Amplification LibraryPreparation—Fusion Method, Life Technologies, Carlsbad, Calif.) usingIon Xpress Barcodes linked to 16S gene primer pairs targeting 5overlapping contigs that covered hyper-variable regions 1-8 sequencingover 90% of the gene ²⁶. DNA from 36 individual fecal samples werestratified into 10 bio-pools created by equal mixing of individualmaterial that then were used as templates for creation of fusion 16Slibraries. Stratification was accomplished for healthy control samplesbased solely upon BMI (above or below 30) creating 2 bio-pools (Table1).

TBI samples were stratified by location, BMI, and whether residence timewas above or below the median time participants lived in either theBakersfield (average of 232 months) or Tideway (average of 241 months)facility creating an additional 8 bio-pools (Table 1). Fusion PCRlibraries, generated in a c1000 thermocycler (Bio-Rad), were purifiedusing QIAquick spin-columns (Qiagen) and quantified using aspectrophotometer (Bio-Rad) before being diluted and then sequenced onan Ion Torrent Personal Genome Machine using 400 base pair read kitstogether with 316 size chips following the manufacturer's instructions(Life Technologies). One TBI bio-pool (BP #3) failed to meet minimalquality metrics and was not analyzed further. Sequencing reads from theother 9 libraries were filtered for quality and binned according to IonXpress barcodes using Ion Torrent Suite software (v5.0.5). Filteredsequencing reads in FASTQ format were normalized using the FASTQ groomertool function in the web-based Galaxy software ²⁷. Next, each barcodedread was trimmed to remove primer sequences and filtered to the expectedsize before being compared to the SILVA 16S rRNA gene database usingBowtie 2 software ^(24,28) Curated reads showed >98% alignment to thedatabase and ranged from 8E5 to 1.2E6 sequences to establish generalevel hit-rates. Where multiple calls to the same genera were made thenumber of hits were added accordingly. These numbers were then convertedto percentage of total to give an overall relative proportion for eachbio-pool.

TABLE 1 Study Cohort Demographics Study Height Weight Time since BP IDGroup # Location Gender Age (cm) (kg) BMI Ambulatory injury (mos) # 1TBI 1A Galveston M 34 182.9 80.6 24.1 N 27 8 2 TBI 2A Galveston M 42193.0 82.8 22.2 N 264 10  3 TBI 3A Galveston M 51 182.9 97.2 29.1 N 5207 4 TBI 4A Galveston M 52 193.0 86.9 23.3 N 177 9 5 TBI 6A Galveston M54 181.6 70.2 21.3 N 400 10  6 TBI 9A Galveston M 54 188.0 95.0 26.9 N423 7 7 TBI 10A  Galveston F 60 167.6 90.5 32.2 N 218 8 8 TBI 7AGalveston M 63 185.4 68.4 19.9 Y 173 NT 9 TBI 8A Galveston M 71 182.972.0 21.5 Y 146 9 Average (SD) 8M/1F 53.4 (10.9) 184.1 (7.6) 82.6 (10.7)24.5 (4.1) 6N/2Y 260.9 (157) 10 TBI B11 Bakersfield M 50 180.34 101.831.3 N 315 3 11 TBI B12 Bakersfield M 54 187.96 98.6 27.9 N 326 3 12 TBIB13 Bakersfield M 36 176.53 87.3 28.0 Y 252 3 13 TBI B14 Bakersfield M57 170.18 77.3 26.7 Y 205 5 14 TBI B15 Bakersfield M 47 180.34 57.7 17.7N 264 6 15 TBI B16 Bakersfield M 39 167.64 76.8 27.3 Y 242 6 16 TBI B17Bakersfield M 62 172.72 70.9 23.8 Y 97 5 17 TBI B18 Bakersfield M 51193.04 105.9 28.4 Y 206 4 18 TBI B19 Bakersfield M 28 180.34 102.3 31.4N 98 4 19 TBI B20 Bakersfield M 39 167.64 72.3 25.7 Y 192 5 20 TBI B21Bakersfield M 36 179.07 106.8 33.3 Y 232 3 21 TBI B22 Bakersfield M 67165.1 83.2 30.5 N 456 3 22 TBI B23 Bakersfield M 60 172.72 69.1 23.2 Y101 5 Average (SD) 13M 48.2 (11.8) 176.4 (8.2) 85.4 (16.2) 27.3 (4.1)8Y/5N 229.7 (101.5) 23 Control 1B Galveston M 33 175.26 74.3 24.2 Y N/A2 24 Control 2B Galveston M 40 193.04 103.5 27.8 Y N/A 2 25 Control 3BGalveston M 49 175.26 119.3 38.8 Y N/A 1 26 Control 4B Galveston M 50182.88 83.3 24.9 Y N/A NT 27 Control 5B Galveston M 57 177.8 83.3 26.3 YN/A 2 28 Control 10B  Galveston F 60 160.02 60.8 23.7 Y N/A 2 29 Control6B Galveston M 61 175.26 103.5 33.7 Y N/A 1 30 Control 7B Galveston M 62175.26 74.3 24.2 Y N/A 2 31 Control 9B Galveston M 66 167.64 65.3 23.2 YN/A 2 Average (SD) 8M/1F 53.1 (11) 175.8 (9.2) 85.3 (19.6) 27.4 (5.4) 9YN/A 32 Control A14 Bakersfield M 24 185.42 85.5 24.9 Y N/A 2 33 ControlA19 Bakersfield M 29 182.88 86.8 26.0 Y N/A NT 34 Control A16Bakersfield F 31 162.56 95.5 36.1 Y N/A 1 35 Control A13 Bakersfield M32 172.72 104.5 35.0 Y N/A 1 36 Control A12 Bakersfield F 34 160.02 85.933.5 Y N/A 1 37 Control A21 Bakersfield M 34 167.64 104.5 37.2 Y N/A 138 Control A11 Bakersfield F 39 162.56 84.1 31.8 Y N/A 1 39 Control A20Bakersfield F 39 170.18 100.0 34.5 Y N/A NT 40 Control A15 Bakersfield M43 177.8 120.5 38.1 Y N/A 1 Average (SD) 5M/4F 33.9 (5.8)* 171.3 (9.2)96.4 (12.2) 33 (4.7)* 9Y N/A Overall TBI 19M 50.3 (11.5) 179.7 (8.8)84.3 (14) 26.2 (4.3) 8Y/11N 242.5 (124.6) Overall Controls 13M/5F 43.1(13.1) 173.6 (9.2) 90.8 (16.8) 30.2 (5.7) 18Y N/A

Average values for each subgroup are shown in bold text with (SDEV)indicated. Biopool (BP) assignments for the second round of IonTorrent-based NGS testing are indicated. Several fecal DNA samples wereof insufficient quality for molecular evaluations and were not tested(NT) in the biopools but were recovered for subsequent qPCR assays.*Bakersfield controls were significantly younger (p<0.05) than any othersubgroup but the overall average age of controls and participants withTBI was not significantly different. There were no differences inaverage height or weight among the subgroups or main cohorts, however,the controls from Bakersfield had a significantly higher average BMIthan the Galvestonians with TBI (p=0.0087) and the combined TBI cohort(p=0.03).

Ion Torrent Sequencing of the tuf gene for Bacteroides and Prevotellagenera also was performed through fusion-primer design using Ion Expressbarcodes. Two sets of primers covering conserved areas flanking thehyper-variable regions were designed based on multiple sequencealignment of 13 Bacteroides and 14 Prevotella species sequences obtainedfrom Genbank. Each primer pair was designed to sequence both DNA strandsof the specified gene region (Primer pair 1, F: CAAACCGCATGTWAAYRTTGGTAC(SEQ ID NO: 193), R: CCRTCCATCTGDGCAGCACC (SEQ ID NO: 194); Primer pair2, F: CGTACTTCTBGCHCGTCAGGT (SEQ ID NO:195), R: ACCTGTAGCHACNGTACCACG(SEQ ID NO:196)) producing coverage of approximately 50% of the tufgene. Data processing and analysis of sequence reads was performed asdescribed above. To identify specific species, NGS-derived tuf sequenceswere compared against a customized database of reference sequences ofBacteroides and Prevotella species sequences obtained from Genbank.

qPCR Evaluation. Based on the sequence data from both approaches andcalculated community profiles, qPCR primers were designed using OligoArchitect (MilliporeSigma; Burlington, Mass.) or obtained from theliterature (Table 2) to quantify organisms or genera of interest. Theseassays were evaluated in silico for specificity to their particulargenera/species and determined to be specific through amplification of asingle PCR product from a bio-pool of the NGS evaluated control and TBIfecal DNAs. PCR products were subsequently cloned and Sanger sequencedauthenticating specificity. These cloned amplimers were then used tocreate high-resolution melt temperatures that served as a furtherconfirmation of identity following SYBR green-based real timeamplification. Each 25 μl qPCR was carried out on each fecal DNA sampleusing: 12.5 μl iQ SYBR green Supermix™ (Bio-Rad), 1 μl of each forwardand reverse (5 μM) primer, 9.5 μl nuclease-free water and 1 μl of DNAtemplate. qPCR was completed in a c1000 thermocycler equipped with aCFX™ reaction module (Model info; Bio-Rad). By this method, all but 2 ofthe fecal samples (controls A19 and A20 failed to meet minimum qualityassessments) were successfully evaluated. Fluorescent signal data wascollected at the end of each extension step. Starting quantity valueswere extrapolated from standard curves of plasmids harboring the PCRtargets run in parallel for each target.

TABLE 2 Custom fecal microbiome qPCR array details. SE SE Q Q ID IDReference TBI Array Primer Forward NO. Reverse NO Target PMIDAkkermansia ATTCTCGGTGTAGCAGTG 1 CTCAGCGTCAGTTAATGTC 2 16s This studyAlistipes obesi GGAAGTATCGTCCGTCTC 3 AAGTGGATTTTCTTGTTCG 4 stLThis study Alistipes GGCTAAAAGCGTACTGAA 5 GTCGATGAAGTAGCTCTC 6DNA polymerase III This study onderdonkii delta Alistipes AAGAATTTCAAGGGTATCATC 7 AGGAGAATCGAGAACAAC 8 EemNRhAT Family This studyputredinis Transporter Alistipes shahii GCTTTCCAAGTATGAGTA 9TATATTCGTCAACGGCAG 10 tu This study Alloiococcus AAAAGAATTGACGGGGAC 11CAAGAGCTGGTAAGGTTC 12 16s This study Anaerotruncus AACAGAGATTGAAGCGGATG13 GGCGGATCATATCAAGGAA 14 DNA polymerase This study colihominis betaAsteroleplasma TACTTCCGGTCTGGGGTG 15 TTCTTCCGCGTATCAGTG 16 16sThis study Bacteroides caccae GCTCTGGTTACTGAACTGGCA 17CAGATCGACCGACATGTGGT 18 DNA polymerae III This study alphaBacteroides clarus  GTATCGCTGTAGGTATGG 19 CCTCAATGTCAATATCGTTAT 20 gyrAThis study Bacteroides ACCACCTCTTCACCGTAA 21 TGTCACTTGCTCTACTTGTAAT 22dnaJ This study congonensis Bacteroides CGGAGACTTGTTAGTTCTTA 23GGTTGTATATCAGGTCGCTTTC 24 dnaJ This study coprocola TCG BacteroidesTGTGGTAACTGCTTATCTG 25 CATTCTGTCTTGGTCTTGA 26 gyrA This studycoprophilus Bacteroides dorei  TAATACGATCATCCGCTAT 27GCTTGTAAACCTACTTTGT 28 recA This study Bacteroides TATCGGCGACATTAGTAT 29GTTGTCTTCGTTGATAGTAA 30 gyrB This study eggerthii Bacteroides faecisGGTAGTCATTGTCAGAGG 31 GTCATCCACTGAATATAATAATTG 32 DNA polymerase IIIThis study epsilon Bacteroides ACGGAAACGGTGTATGAC 33 TCAGTGAGATGCGTAGTC34 gyrB This study finegoldii Bacteroides  GGCGGTCTTCCGGGTAAA 35CACACTTCTGCGGGTCTTTGT 36 gyrB 20643165 fragilis BacteroidesAAGACAATGTGCTTAATGC 37 GTAGGTCGTCAGTTTAGTTA 38 RNA-directed DNAThis study intestinalis polymerase Bacteroides AATTGGCTTACCTGAACAAG 39CCGAATGGAACACCTCTT 40 gyrB This study massiliensis Bacteroides nordiiGGAGATGCGTCAGATAAC 41 GGTCGGTATGTTCAAGTAG 42 DNA polymerase This studyBacteroides ovatus GGAGGATATTATATTCTGCG 43 CCACTGTATGCGTATGAC 44Hypothetical TAA protein (Strain This study ATCC8483) Bacteroides ACAAAGGAATCACCATCAC 45 TTACGCCTTCTTCTGAGT 46 gyrB This study plebiusBacteroides CCGTTATCTGGAATCATCA 47 GATAGCCACTTCGATAGG 48 gyrB This studysalyersiae Bacteroides  GGCGGCAAGATTTATCAG 49 TATCAGCGGTTCCTACTTC 50gyrB This study sartorii Bacteroides GGAAGCCAATATAGACAAGT 51GGGAAATAGCGGAAGAAG 52 gyrB This study stercoris BacteroidesGCAAGGAGATGAATTATATGA 53 CACATTCCGTTCGTTGAT 54 DNA polymerase IIIThis study sterorirosoris CT delta Bacteroides TACAATTGCCACAGTACGGAA 55GCTGACGAACGATGACCATAGTTA 56 a-1-6 mannanase 23068949 thetaiotoamicron CABacteroides TAGCCAATGATAAGAAGATA 57 TCACGATACGCATACCTT 58 gyrAThis study imonensis GAAG Bacteroides AGCAAAGAAGCGAACATT 59GGGAAGTAGCGGAAGAAA 60 gyrB This study uniformis BacteroidesTTATGAAGAAGGAACCACTC 61 GAATAAGGAAACGCTCAGA 62 Coproporphyrinogen This study vulgatus III oxidase Bacteroides GGAGCATATCGTAAGACTA 63TTGAGCCATTGAATAATCG 64 xyn 10A This study xylanisolvens BifidobactetiumGATGACGATTCCACCGAG 65 TACGAGACATAATCCTTGATGC 66 gyrB This study bifidumBifidobacterium CATGAATCTCGACGATCTGAA 67 CAGGCTGGATGTTCTTGG 68DNA polymerase III This study longum gamma and tau BilophilaCCCGCCATTTATGTGAAG 69 CGAAGTAGCACATTTCCA 70 taurine: pyruvate This study wadsworthia aminotransferase Blautia CTGTCATACTTGAGTGCC 71GTCAGTTACCGTCCAGTA 72 16s This study Butytivibrio GACTGCTTTTGAAACTGT 73CCTCCTAATATCTACGCATT 74 16s This study Candida albicans GGACGTTACCGCCGCAAG 75 GCATCGATGAAGAACGCAGC 76 ITS 15680216 CAATCandida glabrata  GAGCAGCAGATTAATAGAG 77 TGTTTGGTAGTGAGTGATA 78 ITSThis study Clostridium leptum CGTAGAGGTTCTGGTAGA 79 GATTTCTTGGTTCGTCATTT80 rpoD This study Clostridium GGAGCAGGTATATGTGGAA 81CGGCGTATTGATATTATTGACA 82 gyrB This study sybiosum CollinsellaAGCATGTGGCTTAATTCG 83 CCTGTATGGGCTCCTCTC 84 16s This study CoprococcusTATATGAATCTGGTTACGGTA 85 TGGCTGATCGGATAAGTA 86 tryp-tRNA ligaseThis study comes T Coprococcus AGACAGGTGATATAGTAAGC 87CGATGTGATTCTTCCAATG 88 DNA gyrase This study eutactus subunit ACross-assembly CAGAAGTACAAACTCCTAAAA 89 GATGACCAATAAACAAGCCATTAGC 90Single stranded 28700235 phage (CPQ 56) AACGTAGAG DNA-binding proteinCross-assembly TGTATAGATGCTGCTGCA 91 CGTTGTTTTCATCTTTATCTTGTC 92DnaG family 28700235 phage (CPQ 64) ACTGTACTC CAT primase DesulfovibrioCCGTAGATATCTGGAGGAAC 93 ACATCTAGCATCCATCGTTTACAGC 94 16s 20940602 ATCAGDialister CCTAGTGTAGCGGTGAAA 95 GTCAGTTTTCGTCCAGAA 96 16s This studyDorea GGGVVTAACCKGGAGGAAG 97 GTTTACGCCATTGTAGCA 98 16s This studyEcoli universal GTGTGATATCTACCCGCT 101 AGAACGGTTTGTGGTTAATCAGGA 100Beta-D- 12401234 TCGC glucuronidase 12401234 EnterococcusAGGAGAGCAACAAGATAT 103  CGTTCCGCCTTCATAAGT 102 gyrB This study faecalisTACA Enterococcus CACGGAGTAGGATCTTCT 105 CACGACGATATTCTTGATAGT 104 gyrBThis study faecium Entercoccus AGAAATTCCAAACGAACTTG 107CAGTGCTCTACCTCCATCATT 106 23s 15707628 universal EubacteriumACAGCCATCATCAATCTC 109 ACTTGTCATCCTCGTATTC 108 gyrA This study rectaleEubacterium TGCTTGCTATTGATAACAT 111 TTCCTTGAATCGCTCCAT 110 gyrAThis study siraeum TGA Eubacterium ATATACAGGAGGCTATGC 113CGTCTATTACAACAATCTTATC 112 ABC Transporter This study siraeum (StrainDSM15702) Faecalibacterium CCCTTCAGTGCCGCAGT 115 GTCGCAGGATGTCAAGAC 11416s 26839545 prausnitzii Fusobacterium CAACCATTACTTTAACTCTAC 117GTTGACTTTACAGAAGGAGATTATG 116 Anti-termination 22009989 nucleatumCATGTTCA TAAAAATC protein nusG Fusobacterium TARGCGGAACTACAAGTG 119CAGTAATCTGTCCAGTAAGC 118 16s This study HaemophilusGGAGTGGGTTGTACCAGAAGT 121 AGGAGGTGATCCAACCGCA 120 16s 9574673 universalAGAT Holdemania AGCGTTATATCGGACTTAC 123 TTCAGCGTAGTGACAATG 122DNA polymerase This study filiformis III gamma Lachnoclostridium CTRGTGTAGCGGTGAAATG 125 ACGTCAGTTACWGTCCAGT 124 16s This studyLactobacillus TGGCCCAATTGATTGATG 127 GCATCTGTTTCCAAATGTTG 126 16sThis study fermentum Lactobacillus CAGACAATCTTTGATTGTT 129GCTTGTTGGTTTGGGCTCTTC 128 165-23s ITS 10856652 reuteri TAG LactobacillusTGCTTGCATCTTGATT 131 GGTTCTTGGATYTATGCGGTATTAG 130 16s 15243071rhamnosus TAATTTTG Lactobacillus ATGGAATTTGGAGATTATGGT 133TGCTGGGTAATGTGCTTT 133 DNA polymerase This study salivarius TT III alphaLactobacillus CTCAAAACTAAACAAAGTTTC 135 CTTGTACACACCGCCCGTCA 134 16s12351242 universal Lactococcus lactis GCTAATAACATCAACACTCA 137TAACTGCTGTCAATCCTT 136 gyrB This study Leptotrichia GGAAAKGTGGGTGGAACTA139 GTTATCTTCATCATCGGCATTC 138 16s This study MegasphaeraAAGAATCGTATCGTCGTTACA 141 ACAAGTCGGCAATGGATT 140 gyrA This studymassiliensis Methanobrevibacter  GAAAGCGGAGGTCCTGAA 143ACTGAAAAACCTCCGCAAAC 142 nif 21070516 smithii OrdotibacterGGCGGAATAAGTTAAGTAGC 145 CAGCGTCAGTTAYRGTCT 144 16s This studyParabacteroides CGGTGACTTATTGATCCTTA 147 AATAGCAGGTTGTATAACAGA 146 dnaJThis study distasonis Parabacteroides TCTACCTGTCCGACTTGT 149 ATCACGCATAACACCTTCA 148 dnaJ This study qoldsteinii ParabacteroidesCAACAATGTGCCGAACCT 151  CGCTTACCTGCTTCTTCA 150 atL This study merdaeParasutterella ATGGCTGACGAAGTAGAT 153  GGCGATAACCTTTCTTGAT 152 dnaJThis study excrementihominis Prevotella copri AGATAGGCAATGTGGAGTAT 155ATCTATCGCATCGCTCTC 154 DNA polymerase This study III alpha Prevotella GAATGAGCCGCACTATAC 157  GAATGAGTTGATAACACTTGAA 156 rpoB This studycorporis Prevotella disiens CCAAAGCAGCACAAATGA 159 CCACCTTATCAAGTTCAGATG 158 polD This study Prevotella TATCCGTGAGCGTATGAA161  TTCGTTCCAAAGAATGAGTTA 160 rpoB This study melaninogenica PrevotellaCAACTATCTTGAGGAGAACC 163  CCTTACGTGCTGCGATAC 162 gyrB This studystercorea Prevotella spp GGGATGCGTCTGATTAGCTTG 165 CTGCACGCTACTTGGCTGGTTC 164 16s 20305015 TT Pseudoflavoni-GACGCCATCATCCTCATC 167  GACAACCTGCTCCAGAAC 166 hsp60 This study fractorcapillosus Ruminococcus GAACGATTGGGACTTCTTA 169  GAGGTATCTTCTCCAAGTC 168DNA polymerase This study bicirculans III alpha RuminococcusGAAGAGCCGAAAATCATC 171  GGTCATCAATACGCAAAT 170 DNA polymerase This studybromii III alpha Ruminococcus AAGAAGAAGATGCGGATCA 173 AAGTGCTGGTTGTGGTAT172 dnaJ This study callidus Ruminococcus TAGCCAAGAAGTATCATCCA 175CACTGTATGCCTCTGTTG 174 dnaJ This study obeum RuminococcusTTTCTGCCTGATGATACG 177 TATTGATATGCTCGGTCTG 176 gyrB This study torquesStreptococcus AATCCAACAAGAACTTTACG 179 CATCAACATCTGCTGGTA 178 ketol-acidThis study salivarius reductoisomerase StreptococcusAGTCGGTGAGGTAACCGTAAG 181 AGGAGGTGATCCAACCGCA 180 16s 9574673 universalSubdoligranulum AACCCATAAATTGCTTTCA 183 ATATCTACGCATTCCACC 182 16sThis study Sulfate-reducing ACSCACTGGAAGCACG 185 GGTGGAGCCGTGCATGTT 184dsrA 17351812 Bacteria - DSCR1 Suffererla CGCGAAAAACCTTACCTAGCC 187GACGTGTGAGGCCCTAGCC 186 16s 22233678 Veillonella GACGAAAGTCTGACGGAG 189CCGATTAACAGAGCTTTACAA 188 16s This study 16s TCCTACGGGAGGCAGCAGT 191GGACTACCAGGGTATCTAATCCTGTT 190 16s 14532224 hGAPDH CAACTACATGGTTTA 193CTCGCTCCTGGAAGATG 192 hGAPDH 15927278 CATGTTC

Amino acid analysis. Postprandial plasma amino acid levels were assessedfor both TBI and control subjects as previously described². In summary,following overnight fasting and consumption of a standardizedmixed-macronutrient meal, 5 ml of blood was drawn via venipuncture fromsubjects approximately 90 minutes after completing the meal. Fresh bloodwas separated by centrifugation at 3,000 RPM for 20 minutes and serumfractions were stored at ˜80° C. (or on dry ice while in transit fromBakersfield to Galveston) until analysis. Serum amino acidconcentrations were determined after protein precipitation using aHitachi L8800 amino acid analyzer (Hitachi, Tokyo, Japan) according tomanufacturer guidelines.

Statistical analysis. Bacterial diversity was assessed by calculatingthe number of observed OTUs and the Shannon diversity index (SDI) basedon 16S rRNA gene compositional analysis. The number of observed OTUsmeasures bacterial richness in a sample, while SDI measures bothrichness and evenness. Between sample diversity, or resemblance, wasassessed with weighted and unweighted UniFrac metrics and plotted usingprincipal coordinate analysis (PCoA) ordination 29 To examine thecontribution of different taxa to diversity and community composition,the relative abundances of taxa at the OTU, genus, and phylum level werecalculated. Diversity data were analyzed using R³⁰. Differences betweenalpha diversity in TBI v. controls were assessed with Wilcoxon rank-sumtests. Differences in beta diversity (Unifrac distance) were assessedusing PERMANOVA. All P-values were adjusted for multiple comparisonswith the FDR algorithm ³¹. MixOmics ³² was implemented in R version3.3³⁰ to determine the correlations between the bacterial relativeabundance and the concentration of selected amino acids³³. MixOmics usedsparse partial least squares regression (sPLS) and was performed incanonical mode with LASSO penalization. Statistical analyses of the qPCRdata were performed using Excel™ (Microsoft Corp., Redmond, Wash.) orPrism (GraphPad, Inc. v7.0e) software packages. Clustering analysis wascompleted using Morpheus web-based software (Broad Institute, Cambridge,Mass.). For comparisons of the qPCR absolute abundance data, statisticalsignificance was determined using Multiple t tests via the Holm-Sidakmethod (Prism). Each qPCR target was analyzed individually, withoutassumption of a consistent SD. A p value of <0.05 was consideredsignificant.

Study Cohort. Fecal samples were collected from a cohort of patientswith chronic, moderate to severe TBI residing in permanent carefacilities located in Galveston, Tex. and Bakersfield, Calif. (n=22) andfrom healthy control subjects (n=18). The control cohort was enrolledfrom the care facilities in an effort to limit potential confoundingfactors, as they shared both environment and ate some of the same mealswith the TBI patients. The demographics of the cohorts are summarized inTable 1. In general, patients from the Texas cohort were minimallymedicated while a more aggressive drug therapy approach was utilized inthe California cohort. All but one of the TBI samples was collected fromparticipants who had not taken any recent antibiotic treatments.Similarly, only one of the controls was actively taking amoxicillin atthe time of fecal sampling. Most of the other medications were oraldietary supplements (e.g., fish oil) or medications related to mooddisorders (e.g., Zoloft).

Individuals in the Galveston program resided in private or semi-privaterooms within one building. Meals were planned and supervised by adietician and plated for the individual. When the individuals went onoutings, they were free to eat ad lib. Individuals in the Bakersfieldprogram lived in one- or two-bedroom apartments within a large apartmentcomplex. They followed a meal planning menu supervised by a dieticianand went to the grocery store to purchase their own food. Meals wereprepared by that individual with help as needed and they were also freeto eat ad lib when on outings.

There were no significant differences between the two main cohorts(overall TBI versus overall control) regarding average age, height,weight or BMI (multiple t test p>0.05). Comparisons of the average timesince TBI injury revealed no differences between the two sites nor werethere differences between a facility cohort and the overall average(p>0.99). Considering each facility subgroup, the Bakersfield controlswere significantly younger (p<0.05) than any other subgroup but theoverall average age of controls and participants with TBI was notsignificantly different. There were no differences in average height orweight among the subgroups, however, the control cohort from Bakersfieldhad a significantly higher average BMI than the Galvestonians with TBI(p=0.0087) and the combined TBI cohort (p=0.03). Because weight/BMIdifferences may be significant contributors to the intestinal microbiomeprofile, the inventors completed all analyses of 16S gene sequencingdata and qPCR outcomes both by comparing the overall TBI versus overallcontrol, and by comparing site-specific control versus site-specific TBIresults. This approach also addressed any potential impact fordifferences in drug therapy regimens between the facilities.

Shifted fecal microbiomes were identified in chronic TBI cohortscompared to controls. Fecal DNA from each person in the cohort wasinitially evaluated for quality prior to subsequent molecularevaluations. Four samples failed to meet quality metrics for amplifiableDNA (16S gene and human targets) and were excluded from the NGS analyses(Table 1; NT). The remaining DNA samples were subjected to 16S rRNA geneV4 sequencing. Inclusion of all available samples from both collectionsites illustrated that the microbiome profiles were significantlydifferent between TBI and controls, both by weighted UniFrac (FIG. 1A;p=0.002) and unweighted UniFrac (FIG. 1B; p=0.005) PCoA analysis.UniFrac is a distance metric that incorporates phylogenetic relatednessof taxa in the analysis. The weighted UniFrac metric considered relativeabundance of bacterial OTUs showing that the overall structure of themicrobial communities was different between groups (FIG. 1A). Theunweighted UniFrac metric evaluated only the presence or absence ofbacterial OTUs and demonstrated that the community structure also wassignificantly different between TBI and controls (FIG. 1B).

To evaluate if the observed differences were maintained when only asingle location was compared, the inventors reran the weighted UniFracPCoA analysis between TBI and matched controls from each site, andconfirmed that significant differences between TBI and controlspersisted in both the Galveston (p=0.002) and Bakersfield (p=0.006)cohorts (not shown). Notably, the overall relative abundance ofbacterial genera from samples collected at each site was comparable, andno single genera was found to be significantly different betweenlocations (not shown); supporting a lack of geographical bias andreducing concerns about the impact of other confounders. Next, theinventors compared alpha diversity and bacterial relative abundancebetween the overall TBI and control groups. The number of observed OTUswas not significantly different between TBI and controls (p=0.21), butthe SDI (richness and evenness) was significantly higher in TBI cohortscompared to controls (p=0.008; FIG. 2).

The data revealed that there were significant shifts even at the phylalevel when the average TBI community structure was compared to thecontrol cohort. The most abundant bacterial phyla were Bacteroidetes andFirmicutes, with the relative abundance of Bacteroidetes significantlyhigher in controls (p=0.002) and the relative abundance of Firmicutessignificantly higher in TBI (p=0.002; FIG. 3A). Also, the relativeabundance of Actinobacteria (p=0.002) and Verrucomicrobia (p=0.038) weresignificantly higher in TBI compared to controls (FIG. 3A). At thefamily level, Prevotellaceae (phylum Bacteroidetes) was significantlymore abundant in controls (p=0.03) and, two unclassified genera ofFirmicutes from the family Ruminococcaceae were significantly higher inTBI (p<0.001; FIG. 3B). The relative abundance of the 3 significantbacterial families was indistinguishable between the two collectionlocations (data not shown).

Because 16S V4 rRNA gene sequencing data are limited to family/genuslevel analyses, the inventors performed metagenomic shotgun sequencingand analyzed the resulting data at the genus and species levels. Theseanalyses corroborated the 16S rRNA gene evaluations confirming thatPrevotella was significantly more abundant in controls (p<0.001), andRuminiclostridium trended toward a greater abundance in TBI (p=0.063).Within the metagenomic data, at the species level, Prevotella stercoreawas significantly higher in controls (p<0.001). However, qPCR assays,described below, targeting two distinct P. stercorea genes showed thatmost samples were negative for this species. Because this organism wasfound in only a few samples from both controls and TBI, the inventorsconcluded that other Prevotella species were likely missed by the NGSanalyses.

To rigorously confirm the differences identified through Illumina 16S V4and metagenomic comparisons, the inventors also employed a novel NGSapproach that utilized Ion Torrent sequencing of five overlappingcontigs created by amplification of the bacterial 16S rRNA gene ³⁴. Thismethod produced bi-directional sequences from >90% of the bacterial 16SrRNA gene including all of the variable regions, but had lowerthroughput relative to Illumina NGS. As a result, the inventors created10 bio-pools of fecal DNA (equivalent bacterial genomic contributions ofthe indicated samples mixed into a single DNA pool), by stratifying thecohorts by BMI, and by time, since TBI (Table 1). Coincidentally, thisalso led to some Bakersfield- and Galveston-specific bio-pools allowingfor more direct comparisons to confirm a lack of collection siteeffects. One of the libraries (representing bio-pool 3) failed to meetminimal quality criteria and was excluded.

Results from the nine successful bio-pools (two control and seven TBI)corroborated much of the Illumina 16S rRNA V4 and metagenomic data withsome exceptions. At the genus level, the control samples had higheraverage relative abundance of Prevotella (88 fold increase over TBIsample average), Clostridium (5.7 fold) and Faecalibacterium (2 fold;Table 3). Relative to the average control abundance, TBI samples hadhigher levels of Akkermansia (21 fold), Anaerotruncus (2 fold),uncultured Christensenellaceae (5.7 fold), Clostridium (5 fold),Collinsella (3 fold), Desulfovibrio (4 fold), Flavonifractor (4 fold),Odoribacter (16 fold), Parabacteroides (2.3 fold), Streptococcus (8.8fold; Table 3). Comparisons between bio-pools also indicated no obviousdifferences associated with collection site or BMI differences.

TABLE 3 Compressed Ion Torrent NGS Results. Control Control TBI TBI TBITBI TBI TBI Genus BMI <30 BMI >30 BP4 BP5 BP6 BP7 BP8 BP9 Akkermansia0.05 0.07 0.051 0.218 1.474 4.845 0.239 Anaerotruncus 0.16 0.57 0.110.043 1.976 1.331 0.286 0.091 Alistipes 2.41 1.99 1.92 0.593 6.010 4.0530.299 1.142 Bacteroides 25.61 17.71 32.77 25.102 1.906 15.509 14.4989.651 Blautia 4.75 1.62 1.91 7.304 2.240 4.193 1.998 3.060Christensenellaceae 0.06 0.57 1.58 0.393 2.971 0.454 0.068 2.851uncultured Clostridium 1.48 0.18 0.05 0.142 0.181 0.368 0.061Collinsella 0.53 1.36 0.263 1.689 2.144 6.686 1.175 Coriobacteriaceae0.21 0.157 0.108 1.019 0.419 0.291 uncultured Desulfovibrio 0.11 0.521.128 0.189 0.153 0.042 Enterorhabdus 0.20 Faecalibacterium 3.05 3.371.71 2.003 1.464 2.076 0.219 2.586 Flavonifractor 0.13 0.07 0.11 0.0420.072 1.270 Lachnospiraceae 3.15 3.05 6.06 3.673 1.323 5.782 5.084 3.221uncultured Lactobacillus 2.96 13.568 0.036 0.049 Odoribacter 0.05 0.060.33 0.439 1.361 Parabacteroides 2.85 1.22 1.30 1.247 2.670 5.235 20.3032.409 Prevotella 8.67 17.09 0.12 0.130 Prevotella 0.252 0.149 unculturedRoseburia 0.42 0.61 0.04 0.469 0.134 0.377 1.007 Ruminococcaceae 2.466.64 4.52 2.342 12.074 8.034 5.232 10.589 uncultured Ruminococcus 7.8610.14 2.68 18.736 4.189 11.405 0.422 1.529 Streptococcus 0.26 4.36 3.2370.296 3.588 Subdoligranulum 1.04 1.15 0.14 0.430 0.471 2.941 0.798 1.518Sutterella 1.41 1.38 3.20 0.327 3.134 0.048 1.630 0.480 TBI Control AvgTBI Avg Fold- Control TBI Genus 10 (SDEV) (SDEV) change detects detectsAkkermansia 0.649 0.05 1.08 (1.7) 20.9 1 7 Anaerotruncus 1.365 0.37(0.3) 0.74 (0.8) 2.0 2 7 Alistipes 1.229 2.20 (0.3) 2.18 (2.1) −1.0 2 7Bacteroides 1.520 21.66 (5.6) 14.42 (11.53) −1.5 2 7 Blautia 3.801 3.18(2.2) 3.50 (1.9) 1.1 2 7 Christensenellaceae 4.313 0.32 (0.2) 1.8 (1.6)5.7 2 7 uncultured Clostridium 0.83 (0.9) 0.16 (0.1) −5.2 2 5Collinsella 5.041 0.94 (0.6) 2.83 (2.5) 3.0 2 6 Coriobacteriaceae 0.1650.21 0.36 (0.3) 1.7 1 6 uncultured Desulfovibrio 0.545 0.11 0.43 (0.4)4.1 1 6 Enterorhabdus 0.233 0.20 0.23 1.1 1 1 Faecalibacterium 1.9213.21 (0.2) 1.71 (0.7) −1.9 2 7 Flavonifractor 0.10 (0.04) 0.37 (0.6) 3.62 4 Lachnospiraceae 2.985 3.1 (0.1) 4.2 (1.8) 1.3 2 7 unculturedLactobacillus 2.96 4.55 (4.6) 1.5 1 3 Odoribacter 1.435 0.06).01) 0.89(0.6) 15.8 2 4 Parabacteroides 0.295 2.04 (1.2) 4.78 (7) 2.3 2 7Prevotella 0.187 12.88 (6) 0.15 (0.04) −87.9 2 3 Prevotella 0.20 (0.1) 02 uncultured Roseburia 0.247 0.51 (0.1) 0.38 (0.3) −1.3 2 6Ruminococcaceae 9.199 4.55 (2.9) 7.43 (3.5) 1.6 2 7 unculturedRuminococcus 7.684 9.00 (1.6) 6.66 (6.5) −1.3 2 7 Streptococcus 0.1740.26 2.33 (2) 8.8 1 5 Subdoligranulum 3.779 1.09 (0.1) 1.44 (1.4) 1.3 27 Sutterella 0.262 1.39 (0.02) 1.30 (1.4) −1.1 2 7

Interrogation of NGS 16S rRNA gene sequences against databases can maskdifferences in individual organisms that may share closely relatedsequences. To better differentiate specific organisms in the fecalsamples and address the P. stercorea outcome noted above, the inventorsdeveloped and completed Ion Torrent NGS of the bacterial tuf gene forthe Bacteroides, Parabacteroides and Prevotella genera, because of thesimilarity of their 16S rRNA gene sequences and their differentialabundance between control and TBI samples. This approach identifiedsequence matches to over 70 species within these genera and furtherconfirmed that Prevotella spp and Bacteroides spp were generally moreabundant in healthy controls compared to TBI, with notable exceptions tothis generalization (Table 4). The tuf gene results revealed that P.copri was the most abundant Prevotella species in the controls (73% oftuf gene sequences representing a 54-fold greater abundance thanmeasured in TBI samples) followed by the previously identified P.stercorea (4.6% of the control detections and 69-fold more abundant thanin TBI samples). Bacteroides species that were more abundant in controlsincluded B. plebius (3.7% of detections and 19-fold greater abundance)and B. massilensis (2.6% and 7-fold). However, this analysis identifiedspecific Bacteroides species that were more abundant in TBI samplesincluding B. unformis (32% of detections and 17-fold higher abundance),B. stercoris (19.6% of detections and 13-fold higher abundance), B.dorei (6.3% of detections and 8-fold more abundant), B. pectinophilus(2.4% and 3.6-fold) and B. vulgatus (6.5% and 2-fold). Finally, thisanalysis revealed species that were proportionally rare, but weredetected in either controls or TBI samples only (Table 4).

TABLE 4 tuf gene NGS to identify species. Avg Avg TBI Control TBI Fold-Genus Species % % change Prevotella copri 73.281 1.352 −54.2 Prevotellastercorea 4.575 0.066 −68.9 Bacteroides plebeius 3.731 0.198 −18.8Bacteroides vulgatus 3.605 6.535 1.8 Bacteroides massiliensis 2.5700.369 −7.0 Bacteroides uniformis 1.930 32.029 16.6 Bacteroides stercoris1.518 19.607 12.9 Bacteroides caccae 1.019 1.256 1.2 Bacteroidesthetaiotaomicron 0.860 1.712 2.0 Bacteroides dorei 0.799 6.335 7.9Bacteroides pectinophilus 0.673 2.415 3.6 Bacteroides salyersiae 0.5110.006 −91.8 Bacteroides ovatus 0.476 1.660 3.5 Bacteroides xylanisolvens0.298 1.603 5.4 Bacteroides fragilis 0.297 1.411 4.8 Bacteroideseggerthii 0.220 0.405 1.8 Bacteroides coprocola 0.207 0.006 −34.9Parabacteroides goldsteinii 0.179 0.474 2.7 Bacteroides finegoldii 0.0990.013 −7.6 Bacteroides intestinalis 0.051 0.617 12.0 Bacteroidessartorii 0.047 0.553 11.7 Parabacteroides distasonis 0.040 0.190 4.8Bacteroides barnesiae 0.031 0.068 2.2 Bacteroides nordii 0.021 0.076 3.6Bacteroides clarus 0.018 0.034 1.9 Bacteroides congonensis 0.017 0.0392.3 Bacteroides helcogenes 0.014 Bacteroides caecimuris 0.008 0.018 2.4Prevotella buccalis 0.007 0.020 2.9 Bacteroides timonensis 0.007 0.003−2.6 Bacteroides stercorirosoris 0.006 0.069 11.0 Bacteroides pyogenes0.006 0.027 4.3 Bacteroides fluxus 0.005 0.012 2.5 Bacteroidesreticulotermitis 0.003 0.003 −1.1 Prevotella oris 0.003 Bacteroidesfaecis 0.002 0.365 156.6 Bacteroides acidifaciens 0.002 0.007 3.0Prevotella conceptionensis 0.002 Bacteroides graminisolvens 0.002Bacteroides cellulosilyticus 0.002 0.008 5.4 Prevotellamultisaccharivorax 0.002 Bacteroides salanitronis 0.001 0.006 7.4Prevotella bryantii 0.001 Prevotella dentalis 0.001 Bacteroides ihuae0.001 Prevotella jejuni 0.001 Prevotella maculosa 0.001 Bacteroidesneonati 0.001 Prevotella bergensis 0.0002 Prevotella saccharolytica0.0002 Prevotella aurantiaca 0.0004 Bacteroides faecichinchillae 0.0004Bacteroides oleiciplenus 0.0004 Prevotella phocaeensis 0.0004Bacteroides luti 0.001 Bacteroides neonati 0.001 Prevotella nigrescens0.001 Bacteroides paurosaccharolyticus 0.001 Prevotella oralis 0.001Prevotella ruminicola 0.001 Bacteroides gallinarum 0.002 Prevotellaintermedia 0.003 Prevotella disiens 0.005 Prevotella ihumii 0.009Prevotella brevis 0.009 Bacteroides helcogenes 0.035 Bacteroidescoprophilus 0.079 Prevotella corporis 0.080

The consistency in the shifted TBI community structure was furtherconfirmed by clustering analysis of qPCR data that provided absoluteabundance (data are summarized in FIGS. 4A to 4C and Table 5). Thesedata were produced from a novel qPCR array the inventors created basedon the combined 16S rRNA and tuf gene NGS data as well as informationfrom the literature. The array allowed simultaneous quantification of 94bacterial (genus or species level), fungal and viral targets as well asquantification of both total 16S and human GAPDH (Table 5). Using thisarray, the inventors evaluated every DNA sample with sufficient qualityallowing direct comparisons of the average absolute abundance profilesin TBI and control samples for these specific targets. This approachalso provided accurate detection rates for each target across thesamples. By this measure, there were no significant differences inaverage control 16 rRNA gene or human GAPDH copy numbers between the TBIand control cohorts (Table 5).

TABLE 5 Absolute abundance of selected bacterial targets based on qPCRanalyses. TBI Controls TBI fold TBI Array Primer Count Average SD CountAverage SD change P value Akkermansia spp 19 5.5E5 5.3E5 11 2.7E5 3.4E52.08 0.082 Alistipes onderdonkii 21 7.2E5 7.8E5 13 3.1E5 4.8E5 2.340.066 Anaerotruncus colihominis 22 5.8E3 7.5E3 15 4.8E3 1.1E4 1.21 0.762Bacteroides caccae 13  4E4 4.8E4 14 7.8E4 6.3E4 0.51 0.087 Bacteroidesdorei 13 5.1E4 8.7E4 7  5E4 7.9E4 1.03 0.969 Bacteroides massiliensis 61.6E3 1.7E3 8 3.2E3 4.7E3 0.51 0.407 Bacteroides plebius 2 4.6E2 1.5E2 4 3E5  4E5 0.002 0.233 Bacteroides sartorii 17  1E4 3.6E4 12 9.2E2  2E310.99 0.315 Bacteroides stercoris 12 1.5E5 2.6E5 10 9.9E4 1.8E5 1.560.561 Bacteroides sterorirosoris 14 9.4E4  3E5 9 5.9E2 1.2E3 158.530.261 Bacteroides thetaiotoamicron 15 2.8E4 3.6E4 10 6.2E3 1.4E4 4.570.044 Bacteroides uniformis 22 8.5E5 1.3E6 16 5.9E5 7.7E5 1.44 0.452Bacteroides vulgatus 10 1.6E5 1.5E5 12 1.2E5 1.5E5 1.25 0.622Bifidobacterium bifidum 7 1.4E5 1.4E5 3 3.6E5 1.4E5 0.39 0.082Clostridium leptum 20  3E4  7E4 13 5.4E3 5.6E3 5.51 0.137 Clostridiumsybiosum 22 3.1E3 5.3E3 14 1.5E3 3.1E3 2.13 0.247 Collinsella spp 211.1E6 9.7E5 16 1.3E6 1.7E6 0.89 0.759 Desulfovibrio spp 21 6.9E4  9E4 15 2E4 2.5E4 3.44 0.028 Faecalibacterium prausnitzii 22  1E6 1.7E6 162.3E6 2.5E6 0.45 0.097 Lactobacillus fermentum 17 2.2E3 8.9E3 11 1.4E23.9E2 15.58 0.357 Lactobacillus reuteri 5 1.3E2 1.8E2 1 10.1 NA 13.02 NALactobacillus salivarius 8 6.6E4 1.2E5 4 1.9E2 2.9E2 341.34 0.171Leptotrichia spp 2 95.5 1.3E2 2  1.6 1 61.22 0.485 Megasphaeramassiliensis 4 7.6E4 1.2E5 2  1.43 1 53157.06 0.297 Ordoribacter spp 181.7E5 2.6E5 11 6.6E4 6.2E4 2.65 0.102 Parabacteroides distasonis 192.1E4  2E4 14 1.7E4 1.7E4 1.22 0.567 Parabacteroides merdae 18 4.9E45.9E4 14 1.5E5 1.9E5 0.33 0.072 Prevotella copri 2 2.3E2 3.3E2 9 2.8E51.5E5 0.001 0.001 Prevotella spp 19 6.9E4 1.5E5 16 2.2E7  3E7 0.0030.009 Prevotella stercorea 2 7.4E2  1E3 2 4.4E4 6.2E4 0.02 0.505Ruminococcus bromii 8 2.9E4 3.9E4 7 1.2E5 1.3E5 0.25 0.132 Streptococcussalivarius 22 3.1E4 5.9E4 15 6.1E3 1.1E4 5.13 0.066 Streptococcus spp 227.7E4 7.5E4 16 1.2E5 1.6E5 0.64 0.314 Sutterella spp 22  1E5 1.6E5 162.7E5 2.6E5 0.37 0.026 16s 22 3.4E8 3.7E8 15 5.5E8 6.6E8 0.63 0.299hGAPDH 16 27.7 57.5 13 45.0  90.7 0.60 0.537

Clustering analysis of the absolute abundance data showed remarkablegrouping of controls and TBI with some exceptions (e.g. 1BTW control and2ATW and 6ATW TBI; FIG. 4A). There were no obvious characteristics inthe available metadata (Table 1) that explained the unexpectedclustering of these samples. From the 94 microbiome targets, the averageabundance of targets identified as different by one of the previousmethods, or found to be significantly different between the cohorts(multiple t test by the Holm-Sidak method, p<0.05), or to have greaterthan 10-fold differences between the cohorts were plotted (FIGS. 4B and4C) and tabulated (Table 5).

PCR results indicated Prevotella spp were detected in 90% of the samplesin each cohort with a 318-fold increase in absolute abundance in thecontrol samples (p=0.009; Table 2). There were no significantdifferences in the Lactobacillus spp levels but L. fermentum and L.salivarius were substantially higher in titer in the TBI samples(average fold change of 15.6 and 341, respectively; FIGS. 4B and 4C andTable 6). Bacterial targets that had significantly higher absoluteabundance in the TBI fecal samples and were commonly found in bothcohorts included Bacteroides sartorii (11 fold), B. sterorirosoris (158fold), Clostridium leptum (5.5 fold), Streptococcus salivarius (5.1fold; Table 5). Two additional targets that were rarely detected butwere substantially higher in TBI samples were Leptotrichia spp (61 fold)and Megasphaera massiliensis (53,000 fold). Bacteroides plebius (630fold) and Prevotella stercorea (59 fold) were also rarely detectedacross both cohorts, but when present, were substantially higher in thecontrol samples.

TABLE 6 Summary data from qPCR analyses TBI Controls Avg Avg TBI foldTBI Array Primer Count abundance SD Count abundance SD changeAkkermansia spp 19 5.5E5 5.3E5 11 2.7E5 3.4E5 2.08 Alistipes obesi 133.5E4 3.6E4 7 1.1E4 1.8E4 3.24 Alistipes onderdonkii 21 7.2E5 7.8E5 133.1E5 4.8E5 2.34 Alistipes putredinis 12 1.8E5 2.3E5 13 3.9E5 4.4E5 0.47Alistipes shahii 13 1.1E4 1.7E4 9 9.9E3 9.7E3 1.13 Alloiococcus spp 22 2E5  2E5 16 1.7E5 1.3E5 1.20 Anaerotruncus colihominis 22 5.8E3 7.5E315 4.8E3 1.1E4 1.21 Bacteroides caccae 13 3.9E4 4.8E4 14 7.8E4 6.3E40.51 Bacteroides dorei 13 5.1E4 8.7E4 7  5E4 7.9E4 1.03 Bacteroideseggerthii 6 5.1E3 6.9E3 3 1.1E4 1.6E4 0.49 Bacteroides faecis 5 6.1E41.2E5 2 3.6E4 1.9E4 1.68 Bacteroides finegoldii 6 9.2E3 8.3E3 5 2.2E42.3E4 0.42 Bacteroides fragilis 12 5.6E4 1.4E5 5 9.9E3 1.5E4 5.62Bacteroides massiliensis 6 1.6E3 1.7E3 8 3.2E3 4.7E3 0.51 Bacteroidesnordii 8 4.4E3 6.3E3 4  8E3 1.5E4 0.55 Bacteroides ovatus 14 3.8E4 6.3E414 4.6E4 5.6E4 0.82 Bacteroides plebius 2 4.6E2 1.5E2 4 2.9E5 3.9E5 0.00Bacteroides salyersiae 4 7.3E4 1.1E5 6 1.1E5 1.8E5 0.66 Bacteroidessartorii 17  1E4 3.6E4 12 9.2E2  2E3 10.99 Bacteroides stercoris 121.5E5 2.6E5 10 9.9E4 1.8E5 1.56 Bacteroides sterorirosoris 14 9.4E4  3E59 5.9E2 1.2E3 158.53 Bacteroides thetaiotoamicron 15 2.8E4 3.6E4 106.2E3 1.4E4 4.57 Bacteroides timonensis 15 4.6E4 1.1E5 11 1.6E4 2.6E42.91 Bacteroides uniformis 22 8.5E5 1.3E6 16 5.9E5 7.7E5 1.44Bacteroides vulgatus 10 1.6E5 1.5E5 12 1.2E5 1.5E5 1.25 Bacteroidesxylanisolvens 13 7.6E5 1.2E6 10 5.1E5 7.5E5 1.47 Bifidobacterium bifidum7 1.4E5 1.4E5 3 3.6E5 1.4E5 0.39 Bifidobacterium longum 17 9.9E4  2E5 122.5E4 2.7E4 3.90 Bilophila wadsworthia 18 7.6E3 5.7E3 15 1.3E4 1.6E40.60 Blautia spp 22 1.5E7 1.9E7 16 9.2E6 7.2E6 1.57 Butyrivibrio spp 211.4E6 1.4E6 15 1.2E6 6.6E5 1.16 Candida albicans 6 4.4E2  6E2 2 1.6E238.3 2.80 Clostridium leptum 20  3E4  7E4 13 5.4E3 5.6E3 5.51Clostridium sybiosum 22 3.1E3 5.3E3 14 1.5E3 3.1E3 2.13 Collinsella spp21 1.1E6 9.7E5 16 1.3E6 1.7E6 0.89 Coprococcus comes 14 3.1E5  4E5 156.3E5  7E5 0.49 Cross-assembly phage (CPQ 56) 12 8.5E5 1.2E6 5 1.5E6 3E6 0.55 Cross-assembly phage (CPQ 64) 10  5E6 4.6E6 4 8.1E6 1.3E7 0.62Desulfovibrio spp 21 6.9E4  9E4 15  2E4 2.5E4 3.44 Dialister spp 221.1E5 1.3E5 16 3.7E5 7.9E5 0.30 Dorea spp 22 2.7E6 2.6E6 16 2.7E6 1.8E61.00 E. coli 10 6.3E3 1.6E4 9 9.3E4 2.7E5 0.07 Entercoccus spp 22 4.2E41.6E5 16  3E4 9.2E4 1.40 Enterococcus faecalis 10 1.8E2 5.1E2 4 3.6E24.2E2 0.50 Eubacterium rectale 16 5.3E5 1.1E6 15 9.4E5 1.1E6 0.56Eubacterium siraeum 17 9.4E4 3.6E5 8 1.1E4 1.5E4 8.93 Eubacteriumsiraeum 15 2.2E2 3.8E2 5 5.9E3 1.3E4 0.04 Faecalibacterium prausnitzii22  1E6 1.7E6 16 2.3E6 2.5E6 0.45 Fusobacterium nucleatum 17 83   2.8E212 33.2 76.2 2.51 Fusobacterium spp 20 6.6E2 2.1E3 16 3.6E2 7.2E2 1.83Haemophilus universal 22 4.1E3  1E4 16 5.7E3 8.4E3 0.72 Holdemaniafiliformis 10  7E3 5.4E3 8 4.5E3 6.8E3 1.55 Lachnoclostridium spp 223.7E6  3E6 16 4.4E6 2.5E6 0.84 Lactobacillus fermentum 17 2.2E3 8.9E3 111.4E2 3.9E2 15.58 Lactobacillus rhamnosus 20 1.1E2 3.6E2 10 57.6 9.6E11.92 Lactobacillus salivarius 8 6.6E4 1.2E5 4 1.9E2 2.9E2 341.34Lactobacillus spp 20 6.3E4 2.5E5 15 4.6E4 1.7E5 1.38 Lactococcus lactis22 5.9E3  2E4 16 5.8E3 1.6E4 1.02 Megasphaera massiliensis 4 7.6E4 1.2E52  1.4 1  53157.06 Methanobrevibacter smithii 10 5.6E4 7.4E4 7 2.5E43.8E4 2.24 Ordoribacter spp 18 1.7E5 2.6E5 11 6.6E4 6.2E4 2.65Parabacteroides distasonis 19 2.1E4 1.9E4 14 1.7E4 1.7E4 1.22Parabacteroides merdae 18 4.9E4 5.9E4 14 1.5E5 1.9E5 0.33 Parasutterellaexcrementihominis 10 3.3E3 6.5E3 12 2.5E3 3.9E3 1.32 Prevotella copri 22.3E2 3.3E2 9 2.8E5 1.5E5 0.00 Prevotella corporis 6 5.4E2 9.6E2 5  1E21.3E2 5.40 Prevotella disiens 5 1.3E2 1.1E2 3 29.7 34.1 4.52 Prevotellaspp 19 6.9E4 1.5E5 16 2.2E7  3E7 0.002 Prevotella stercorea 2 7.4E2  1E38 4.4E4 6.2E4 0.02 Pseudoflavonifractor capillosus 21  2E3 2.6E3 162.6E3 3.6E3 0.77 Ruminococcus bicirculans 13  5E5 5.1E5 8 4.6E5  7E51.08 Ruminococcus bromii 8 2.9E4 3.9E4 7 1.2E5 1.3E5 0.25 Ruminococcusobeum 14 6.9E4 7.1E4 15 5.7E4 7.7E4 1.21 Ruminococcus torques 21 2.3E35.3E3 13 2.7E2 5.2E2 8.58 Streptococcus salivarius 22 3.1E4 5.9E4 156.1E3 1.1E4 5.13 Streptococcus spp 22 7.7E4 7.5E4 16 1.2E5 1.6E5 0.64Subdoligranulum spp 19 6.2E4 9.7E4 13 7.6E4 1.2E5 0.81 Sulfate-reducingBacteria - DSCR1 20  2E4 2.1E4 15 1.7E4 1.6E4 1.17 Sutterella spp 22 1E5 1.6E5 16 2.7E5 2.6E5 0.37 Veillonella spp 22 2.4E3 4.3E3 14 2.4E46.1E4 0.10 16s 22 3.4E8 3.7E8 15 5.5E8 6.6E8 0.63 hGAPDH 16 27.7 5.8E113 45.9 90.7 0.60

Correlations between specific microbes and amino acid levels andbiosynthesis potential. The inventors previously reported one of thelong-term impacts of moderate/severe TBI was altered amino acid levelspost-meal ². Such alterations could lead to or be caused by alteredmucosal microenvironments contributing to the creation and/ormaintenance of a shifted microbiome. Subjects in both cohorts were askedto fast and then were provided a standardized meal followed by a blooddraw 90 minutes after the meal was consumed. Consistent with theprevious report, the TBI patients had significant reductions in theconcentration of a number of amino acids as shown in Table 7. These datawere correlated with the metagenomic data to reveal a positivecorrelation between the relative abundance of Prevotella spp. and allamino acids.

The present invention includes providing or eliminating from the gutflora of a TBI patient one or more of the bacterial listed in Tables 3,4, 5 and/or 6, as the case may be, to bring the gut flora to alignmentwith a normal gut flora. For example, if the bacteria are listed asbeing found in higher amounts in TBI patients, then those bacteria canbe eliminated from the gut flora.

Conversely, if the TBI patient lacks certain bacteria (or have a lowrelative presence of the bacteria in their gut flora), then the gutflora can be supplemented with the missing or reduced bacteria to,again, bring them toward a more normal distribution of flora found innormal individuals (subject that do not have TBI and/or another diseaseor condition).

TABLE 7 Serum Amino Acid Concentrations in TBI and control cohortsfollowing a standardized meal. TBI TBI Control Control P Amino Acid AvgSDEV Avg SDEV value L-Threonine 81.8 23.23 104.2 35.41 0.021L-Tryptophan 36.3 13.54 50.6 13.59 0.002 L-Methionine 17.0 4.32 19.85.11 0.065 1-Methyl-L-histidine 6.8 4.31 9.7 6.45 0.093L-a-Amino-n-butyric Acid 10.0 2.67 13.2 5.01 0.014 L-alpha-AminoadipicAcid 0.7 0.52 1.1 0.75 0.093 L-Sarcosine 34.1 30.17 56.1 27.74 0.023tryptophan/LNAA 0.06 0.02 0.08 0.02 0.068 tryptophan/BCAA 0.12 0.04 0.150.04 0.063

Values shown are concentration in uM except for the last two rows thatshow the ratio of the indicated amino acids for each cohort. P value wascalculated by a Student's T test with p<0.05 considered significant asindicated by bolded text.

In contrast, the relative abundance of the two unclassifiedRuminococcaceae spp. were generally negatively correlated with relativelevels of the amino acids. Tryptophan:BCAA ratio, tryptophan:LNAA ratio,L-tryptophan, B-alanine and alanine showed the strongest positivecorrelations with Prevotella (FIG. 5). Using the one minus Pearsoncorrelation algorithm for the absolute quantities (FIG. 4) the inventorseliminated those samples that failed to group with the proper cohortproducing a data set with those samples that clustered exclusively tocontrol or TBI cohorts (most similar to the average group profile).Similar statistical differences were identified for both L-Tryptophan(Control, 1.67 vs TBI, 1.54; p=0.025) and also for L-Sarcosine (Control,1.66 vs TBI, 1.00; p=0.029) by unpaired t testing, corroborating themetagenomic correlations.

Metagenomic data from individual samples also were used to identify KEGGmodules related to amino acid metabolism and fatty acid biosynthesis(summarized in Table 8). The results showed that the Akkermansia,Bacteroides and Streptococcus genera as well as a number of unclassifiedmetagenomes that were more abundant in TBI samples, carriedsignificantly higher levels of amino acid biosynthesis capabilitiesrelative to the control samples (Table 8). Although not significant, thepresence of Roseburia contributed to the overabundance of amino acidbiosynthetic pathways in the TBI samples. The Bacteroides levels were ofmixed results between the cohorts similar to the NGS and PCR resultsshowing specific species were associated with TBI or controls. At theBacteroides genus level, control communities carried significantlyhigher levels of biosynthetic machinery for isoleucine, leucine, lysineand serine (Table 8). Considering the compiled KEGG module data,metabolic machinery for cysteine, histidine, isoleucine, leucine,lysine, methionine, ornithine, proline, serine, tryptophan, tyrosine andvaline were identified as more abundant in the TBI fecal communitiescompared to controls. Because of the potent signaling and neurologicalfunctions of short chain fatty acids the inventors also noted thatAkkermansia, Roseburia and unclassified metagenomes also significantlyincreased the machinery for initiation and elongation of fatty acids(Table 8).

TABLE 8 KEGG pathway modules identified as significantly over or underrepresented in TBI fecal samples compared to the control cohort. ControlTBI KEGG Control AVG TBI AVG Detect Detect Module Description Genus(SEM) (SEM) n = 16 n = 20 T-test M00021 Cysteine biosynthesis,Streptococcus 54 (23) 198 (52) 19% 65% 0.024 serine => cysteineMetagenome 11 (3) 23 (5) 81% 60% 0.046 M00026 Histidine biosynthesis,Metagenome 18158 (1780) 26413 (2367) 100%  100%  0.009 PRPP => histidineM00570 Isoleucine biosynthesis, Akkermansia 470 (189) 2001 (641) 44% 70%0.036 threonine => 2-oxobutanoate => Metagenome 19510 (2088) 27425(2194) 100%  100%  0.013 isoleucine M00535 Isoleucine biosynthesis,Akkermansia 101 (40) 536 (169) 56% 70% 0.025 pyruvate => 2-oxobutanoateBacteroides 266 (93) 31 (7) 56% 20% 0.035 M00432 Leucine biosynthesis,Akkermansia 193 (82) 1078 (323) 56% 70% 0.018 2-oxoisovalerate =>Bacteroides 265 (93) 37 (18) 56% 10% 0.04 2-oxoisocaproate Metagenome10063 (978) 14103 (1485) 100%  100%  0.03 M00016 Lysine biosynthesis,Akkermansia 277 (121) 1278 (443) 38% 60% 0.049 succinyl-DAP pathway,Bacteroides 775 (218) 77 (31) 31% 10% 0.032 aspartate => lysine M00525Lysine biosynthesis, acetyl- Akkermansia 277 (121) 1278 (443) 38% 60%0.049 DAP pathway, Bacteroides 775 (218) 77 (31) 31% 10% 0.032 aspartate=> lysine M00526 Lysine biosynthesis, DAP Akkermansia 279 (111) 1216(409) 44% 70% 0.043 dehydrogenase pathway, Bacteroides 775 (218) 77 (31)31% 10% 0.032 aspartate => lysine M00527 Lysine biosynthesis, DAPBacteroides 754 (281) 281 (24) 69% 25% 0.036 aminotransferase pathway,aspartate => lysine M00035 Methionine degradation Metagenome 5677 (435)8174 (900) 100%  100%  0.019 M00028 Ornithine biosynthesis, Akkermansia286 (123) 1297 (408) 50% 70% 0.031 glutamate => ornithine Metagenome11665 (1164) 15684 (1530) 100%  100%  0.044 M00015 Proline biosynthesis,Akkermansia 54 (27) 267 (80) 50% 70% 0.023 glutamate => prolineStreptococcus 49 (23) 186 (57) 31% 65% 0.042 M00020 Serine biosynthesis,Bacteroides 7 (1) 3 (1) 13% 10% 0.03 glycerate-3P => serine Metagenome17 (3) 51 (12) 56% 60% 0.018 M00023 Tryptophan biosynthesis, Akkermansia366 (165) 1841 (609) 50% 70% 0.034 chorismate =>tryptophan Bacteroides257 (236) 1353 (374) 13% 40% 0.045 Metagenome 12547 (1222) 18751 (2119)100%  100%  0.017 M00025 Tyrosine biosynthesis, Metagenome 4013 (710)6488 (941) 69% 85% 0.046 chorismate => tyrosine M00019 Valine/isoleucineMetagenome 17117 (1793) 24016 (2010) 100%  100%  0.015 biosynthesis,pyruvate => valine/2-oxobutanoate => isoleucine M00083.3 Fatty acidbiosynthesis, Akkermansia 256 (99) 1140 (325) 50% 70% 0.02 elongationRoseburia 134 (46) 594 (201) 69% 70% 0.04 M00082 Fatty acidbiosynthesis, Metagenome 9967 (801) 14390 (1763) 100%  100%  0.03initiation

Patients with chronic traumatic brain injury (TBI) requiring long-term,permanent care suffer a complex and varied set of clinical symptoms andcomorbidities that persist for many years beyond the acute brain injuryincluding hypoaminoacidemia and altered pituitary function. The resultsfrom this novel, two-site, clinical investigation unequivocallyillustrates that chronic TBI patients exhibit different fecal microbiomecommunity structures compared to controls. These differences persistedand became more defined after rigorous secondary targeted analyses incombination with group stratification and confound removal. The absenceor reduction of Prevotella spp. and Bacteroidies spp., and higherabundance of Ruminococcaceae spp. in chronic TBI compared to controlsoffers immediate therapeutic targets for further investigation inminimizing patient morbidity.

Notably, these results demonstrate that altered bacterial communitiesdirectly affect TBI. By way of explanation, and not a limitation of thepresent invention, these shifts in community structure are likelycreated and maintained by and also contribute to an altered intestinalmucosa that creates a selective microenvironment in a perpetuatingcycle.

The decreased post-meal amino acid levels of the TBI patients in thecohorts show that the intestinal microenvironment would also havealtered amino acid metabolism. Varied abundance of Prevotella copri andBacteroides vulgatus have previously been associated with increasedserum amino acid concentrations especially the branch chain aminoacids³⁵. The present findings show that P. copri was the most abundantPrevotella in the control cohort but this species was significantlyreduced or absent in the participants with TBI. However, B. vulgatus wasrelatively uncommon in the cohorts and, when present, was more abundantin the TBI samples. Bacteria in the Ruminococcaceae family have beenpreviously associated with amino acid deficient microenvironments ³⁶ andwere higher in the fecal samples from TBI patients in the cohort. KEGGanalyses, confirmed that the bacterial shifts occurring in TBI patientsencoded higher levels of necessary machinery for biosynthesis of severalamino acids. These community shifts also may contribute to alteredinflammation that was detected² and, through production of differentmetabolites including short chain fatty acids, would further disruptnormal function of the autonomic nerves (e.g. vagal) altering intestinalcontractility³⁷⁻³⁹. Reduced gut motility also disturbs the microflorabalance and promotes small intestinal bacterial overgrowth contributingto dysbiosis⁴⁰. The present invention proves that the process based onthe combined results in these two geographically distinct cohorts andemphasizes the need for careful cellular and molecular analyses of theTBI intestinal mucosa for treatment.

These studies were completed in individuals that suffered moderate tosevere TBI leading to disabilities requiring full-time supportive care.To address the impact of residence in a care facility the inventorsrecruited from two distinct communities that offered distinct lifestylesand diets. Even in the small TBI cohorts from the two locations theinventors identified significant changes in specific organisms that weresupported and confirmed through multiple NGS approaches and subsequentqPCR analyses. The fecal microbiome profiles the inventors obtained fromthe controls were typical of healthy communities with greater diversitythan was observed in the TBI samples^(12,13). Stratification of the datausing participant metadata did not reveal any obvious confounder beyondTBI. Importantly, the lack of distinction between the two geographicsites where different food, environment and even medication approachesdemonstrate the differences between the control and TBI cohorts. Theinventors' rigorous approaches, each confirming the last, haveidentified a number biomarker organisms in fecal samples with alteredabundance supporting the development of targeted qPCR panels for lessexpensive, higher throughput analyses of larger cohorts that are nowbeing performed.

Consistent with other chronic inflammatory conditions including obesity,the fecal microbiome of chronic TBI patients demonstrated higherabundance of Firmicutes and decreased abundance of Bacteriodetescompared to controls. Within the Bacteroidetes phylum, TBI fecalcommunities showed a loss of Prevotella spp. compared to controls.Despite the development and utilization of a tuf gene targeted NGSapproach the inventors were unable to account for all of the detectedPrevotella sequences at the species level. By several approaches it wasclear that Prevotella spp. were very common in both cohorts (over 90% ofthe individual samples were positive) but the P. copri and P. stercoreaspecies that showed significantly lower abundance in TBI samples werenot as common. Interestingly, expansion of P. copri and P. stercorea inmicrobiome communities has been associated with localized systemicdisease including periodontitis, rheumatoid arthritis, bacterialvaginosis and other chronic inflammatory conditions⁴¹. By way ofexplanation, and in no way a limitation of the present invention, it issuggested that, mechanistically, overly abundant Prevotella spp. havebeen linked to function of T helper type 17 (Th17) cells that can bedirectly causative for types of inflammation⁴². Studies of individualswith non-alcoholic fatty liver disease have suggested that Prevotellaspp. is also significantly reduced relative to controls⁴³. This samestudy also identified that the Alistipes genus was significantly reducedwhile Anaerobacter and Streptococcus genera were increased. The groupconcluded that such community changes led to alterations in microvilliand intestinal barrier integrity (caused by reductions of tightjunctions) consistent with impacts on GI function ⁴³.

Similar disruption of intestinal integrity has been described for TBIoutcomes, but the mechanisms remain unknown. Following TBI in animalmodels and in some clinical studies, expression of proteins associatedwith tight junctions including ZO-1 and occludin are significantlydecreased as are anatomical aspects of the GI tract¹¹. Short-termevaluations of the intestinal microbiome following TBI showed quickchanges in the community profiles in experimental animals. Theinventors' findings are the first to examine and report the long-termoutcomes of TBI on human intestinal communities including impacts onamino acid levels and chronic inflammation².

The inventors observed a significant increase in abundance ofRuminococcaceae spp. in TBI patients that were, on average, 20 yearspost-injury. Although total Bacteroides spp. was higher in the controls,thorough tuf gene analysis and subsequent species-specific PCRapproaches clarified that some specific Bacteroides species were moreabundant in TBI fecal communities. Specifically, three very commonlydetected Bacteroides genus members, B. uniformis (detected in all fecalsamples), B. stercoris and B. thetaiotoamicron (both found in more thanhalf of all samples) were more abundant in TBI by both tufgene and qPCRanalyses.

Finally, the PCR analyses also revealed that Sutterella spp., that weredetected in every sample, were significantly less abundant in TBIsamples than controls. Recent work with microbiomes transplanted frompatients with multiple sclerosis (MS) to mice confirmed decreasedSutterella spp. was associated with MS ⁴⁶. In previous studies, higherSutterella spp. was associated with reduced development of autoimmuneencephalomyelitis in mice ⁴⁷ and better outcomes for individuals withinflammatory bowel disease ⁴⁸. However, a recent study involving humanfecal microbiome transplant (FMT) treating ulcerative colitis indicatedthat enrichment of Sutterella wadsworthensis was associated with poortreatment outcomes ⁴⁹. In this same study Roseburia inulivorans waspositively associated with successful FMT treatment in concert withincreased short chain fatty acid synthesis ⁴⁹.

Utilizing the reconstruction of metabolic pathways from metagenomicdata, the relative abundance of specific metabolic pathways wasevaluated based on the composition of fecal microbiota communities.These data illustrated the selection of bacterial communities that hadgreater capability of biosynthesis of selected amino acids. In additionto critical essential amino acids, sequences encoding synthesismachinery for a number of non-essential amino acids were significantlymore abundant in the TBI samples. Fewer KEGG modules related to fattyacid biosynthesis were significantly associated, but it is notable thatthe Akkermansia spp. and Roseburia spp. also led to increased fatty acidbiosynthesis capability in the TBI communities.

The data generated in this novel, two-site human clinical studyinvolving chronic TBI and control subjects demonstrates an alteredintestinal mucosa lacking available amino acids and fatty acids leadingto enrichment of bacterial types that carry the necessary metabolicmachinery to address this deficit. Although postprandial nutritionalabsorption influences GH secretion, the nutrients responsible forregulating this secretion are not clear ⁵⁰. The enterocytes lining thesmall intestinal tract have rapid turnover and are highly metabolic,scavenging a large proportion of dietary amino acids. These results showthat the chronic and long-lasting effects in these TBI patients are notquickly overcome and can lead to comorbidities long after the initialinjury. The hypoaminoacidemia observed in TBI patients helps explain thesubsequent reduction in growth hormone function and other pituitaryissues seen in many TBI patients. Thus, these results demonstrate thatthe injury-based disruption of intestinal metabolism in TBI patients, inaddition to alterations in nutrient utilization by the microbiota,likely contributed to the altered amino acid profiles observed by thepresent inventors in TBI patients ². The cascade of sequelae can betargeted with multiple treatment methods, including fecal microbiotatransplant (FMT), oral microbiota transplant, colonoscope microbiotatransplant, and the like.

Thus, the inventors identified novel, therapeutically relevantbiomarkers that can be used to treat symptomatic, chronic TBI patients,and offering clinically meaningful treatment options for TBI-relatedcomorbidities. Given the complexity of the impact on both the brain,CNS, immune, metabolic, inflammatory, pituitary, and intestinalmicrobiome, the results herein yielded both therapeutic and mechanisticinsights into TBI. Notably, the results show that supplementation orreplacement of the dysbiotic intestinal community via, e.g., fecalmicrobiota transplant (FMT) can be used to treat TBI patients and itsassociated comorbidities.

The present invention can be provided in a wide variety of modalities.For example, the patient can be given a bacterial composition thatmodified the flora about from the TBI flora to a normal flora, before,during, or after undergoing a colon cleanse treatment. In one example,the bacterial composition can be delivered to the patient bycolonoscope. Further, the bacterial composition does not necessarilyneed to come from a fecal transplant, rather, the bacteria can be labgrown and even customized to address specific needs of the patient.

The inventors of the present invention sought to determine whetherspecific gut microbiota and/or microbial metabolites are associated withbrain injury associated fatigue and/or altered cognition.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

To aid the Patent Office, and any readers of any patent issued on thisapplication in interpreting the claims appended hereto, applicants wishto note that they do not intend any of the appended claims to invokeparagraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), orequivalent, as it exists on the date of filing hereof unless the words“means for” or “step for” are explicitly used in the particular claim.

For each of the claims, each dependent claim can depend both from theindependent claim and from each of the prior dependent claims for eachand every claim so long as the prior claim provides a proper antecedentbasis for a claim term or element.

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What is claimed is:
 1. A method of identifying a patient with a braininjury associated fatigue or altered cognition (BIAFAC) that willbenefit from a probiotic bacteria treatment comprising: identifying ahuman patient with a brain injury associated fatigue or alteredcognition; obtaining a biological sample from the patient that comprisesgut intestinal flora; and determining whether the gut intestinal florain the biological sample comprises a decrease in the presence ofprobiotic bacteria selected from at least one of Prevotella spp orBacteroidies spp bacteria, when compared to a normal human sample. 2.The method of claim 1, further comprising treating the patient with acomposition comprising one or more probiotic bacteria selected from atleast one of: Prevotella spp or Bacteroidies spp, or one or more agentsthat increase the amount of Prevotella spp or Bacteroidies spp in anintestinal flora of the patient, wherein a composition comprises anamount effective to reduce or eliminate the brain injury associatedfatigue or altered cognition.
 3. The method of claim 1, furthercomprising providing the patient with a fecal transplant comprisingPrevotella spp or Bacteroidies spp.
 4. The method of claim 1, whereinthe biological sample is a fecal sample.
 5. The method of claim 1,wherein a presence of a probiotic bacteria is determined by at least oneof metagenomic shotgun sequencing of bacteria, quantitative PCR assaysor metabolic LC-MS analysis of bacterial metabolites.
 6. The method ofclaim 1, further comprising the step of providing the patient with anagent that reduces or eliminates Ruminococcaceae genus bacteria.
 7. Themethod of claim 6, wherein the agent that reduces or eliminatesRuminococcaceae genus bacteria is selected from an amino acid mixture,an antibacterial agent, a bacteriophage, or an antimicrobial CRISP-Cassystem agent.
 8. The method of claim 1, wherein the human patient hasbrain injury that is chronic, mild, or undiagnosed, or is caused bystroke, hemorrhage, surgery, or radiation.
 9. The method of claim 1,wherein a composition is provided to the patient that further comprisesan amino acid mixture that promotes a growth of Prevotella spp orBacteroidies spp.
 10. The method of claim 1, wherein a composition isprovided to the patient further comprises an amino acid mixture thatreduces or eliminates Ruminococcaceae genus bacteria.
 11. The method ofclaim 1, wherein the patient did not receive a concurrent antibiotic ora probiotic therapy.
 12. The method of claim 1, wherein the one or morebacteria is listed in Tables 3, 4, 5 and
 6. 13. The method of claim 1,further comprising providing the human patient a bacterial compositionthat is modified to correct a TBI flora to a normal flora is providedbefore, during, or after undergoing a colon cleanse treatment, deliveredby colonoscope, wherein the bacterial composition is not a fecaltransplant, or wherein bacteria are lab-grown, or wherein bacteria arelab-grown and customized to specifically modify a TBI flora of aspecific patient.
 14. A method of treating a human patient with a braininjury associated fatigue or altered cognition (BIAFAC) that willbenefit from a probiotic bacteria treatment comprising: identifying ahuman patient with a brain injury associated fatigue or alteredcognition; obtaining a biological sample from the human patient thatcomprises gut intestinal flora; and determining whether the gutintestinal flora in the biological sample comprises a decrease in thepresence of probiotic bacteria selected from at least one of Prevotellaspp or Bacteroidies spp bacteria, when compared to a normal humansample; and providing the human patient with a composition comprisingone or more probiotic bacteria selected from at least one of: Prevotellaspp or Bacteroidies spp, in an effective amount sufficient to reduce oreliminate the brain injury associated fatigue or altered cognition. 15.The method of claim 14, wherein the composition comprises one or moreprobiotic bacteria selected from at least one of: Prevotella spp orBacteroidies spp, or one or more agents that increase the amount ofPrevotella spp or Bacteroidies spp in an intestinal flora of thepatient, wherein a composition comprises an amount effective to reduceor eliminate the brain injury associated fatigue or altered cognition.16. The method of claim 14, wherein the composition comprises a fecaltransplant comprising Prevotella spp or Bacteroidies spp.
 17. The methodof claim 14, wherein a presence of a probiotic bacteria is determined byat least one of metagenomic shotgun sequencing of bacteria, quantitativePCR assays or metabolic LC-MS analysis of bacterial metabolites.
 18. Themethod of claim 14, further comprising the step of providing the patientwith an agent that reduces or eliminates Ruminococcaceae genus bacteriaselected from an amino acid mixture, an antibacterial agent, abacteriophage, or an antimicrobial CRISP-Cas system agent.
 19. Themethod of claim 14, wherein the human patient has brain injury that ischronic, mild, or undiagnosed, or is caused by stroke, hemorrhage,surgery, or radiation.
 20. The method of claim 14, wherein thecomposition is provided to the patient that further comprises an aminoacid mixture that promotes a growth of Prevotella spp. or Bacteroidiesspp., and an amino acid mixture that reduces or eliminatesRuminococcaceae genus bacteria.
 21. A method of treating brain injuryassociated fatigue or altered cognition (BIAFAC) in a human patientcomprising: identifying a human patient in need of treatment for a braininjury associated fatigue or altered cognition associated with analtered intestinal flora; and providing the human patient with acomposition comprising at least one of: a Prevotella spp or aBacteroidies spp bacteria, or one or more agents that increase an amountof the Prevotella spp or the Bacteroidies spp bacteria in an intestinalflora of the human patient to reduce or eliminate the brain injuryassociated fatigue or altered cognition.
 22. A blood test assay thatdetermines a biological profile associated with a bacterial flora thatexacerbates a brain injury associated fatigue or altered cognition fortesting a fecal sample from a patient that compares the biologicalprofile from the patient to a database of a normal biological profile,wherein the bacterial flora detected is selected from Prevotella spp,Bacteroidies sp, or Ruminococcaceae genus bacteria.
 23. A kit forscreening a patient for a brain injury associated fatigue or alteredcognition of a patient and choosing a proper treatment based on thescreening, the kit comprising: a screening tool that screens the patientfor a risk of brain injury associated fatigue or altered cognition, thescreening tool comprising a questionnaire and further comprising ascreening score system that determines the risk of the patient based ona cumulative point total from answers to the questionnaire; a diagnostictest that categorizes the brain injury associated fatigue or alteredcognition based on the cumulative point total from the questionnaire; afecal sample collection container for determining a bacterial flora in afecal sample of the patient; instructions for implementing the braininjury associated fatigue or altered cognition treatment; and a braininjury associated fatigue or altered cognition treatment compositioncomprising one or more probiotic bacteria selected from at least one of:Prevotella spp or Bacteroidies spp, or one or more agents that increasethe amount of Prevotella spp or Bacteroidies spp in an intestinal floraof the patient, wherein the composition comprises an amount effective toreduce or eliminate the brain injury associated fatigue or alteredcognition.
 24. A method of diagnosing and treating a patient for braininjury associated fatigue or altered cognition, the method comprising:using an algorithm implemented in a computer program to: screen apatient for a brain injury associated fatigue or altered cognition riskby scoring a questionnaire based on answers from the patient to thequestionnaire, the scoring providing a cumulative point total, anddetermining the brain injury associated fatigue or altered cognition ofthe patient based on the cumulative point total from the questionnaire;and choosing a proper brain injury associated fatigue or alteredcognition treatment product if the brain injury associated fatigue oraltered cognition risk exceeds a threshold, the brain injury associatedfatigue or altered cognition treatment product comprising a compositioncomprising one or more probiotic bacteria selected from at least one of:Prevotella spp or Bacteroidies spp, or one or more agents that increasethe amount of Prevotella spp or Bacteroidies spp in an intestinal floraof the patient, wherein the composition comprises an amount effective toreduce or eliminate the brain injury associated fatigue or alteredcognition.